Tackling a heavy race programme in the run-up to the Athens Games, the Ukrainian defends three titles she won at the 2002 European championships in Berlin.
A virtuoso in swimming's toughest events, she defends her 400 individual medley crown on Monday, the 200 medley on Wednesday and Thursday and the 400 freestyle in the final session next Sunday when she is also expected to contest the 200 butterfly final.
Hungary's Eva Risztov, quadruple silver medallist in Berlin, is Klochkova's leading rival.
Emiliano Brembilla begins the defence of his 400 metres freestyle title in the opening event in the M-86 pool.
His main challengers will be fellow Italian Massimiliano Rosolino and Romania's Dragos Coman, respectively silver and bronze medallists in Berlin, and hugely promising Russian distance swimmer Yuri Prilukov.
Four years ago, when the European championships were held in Helsinki a little over two months before the Olympics, Brembilla won the 400 from Coman, with Rosolino fourth.
However, Rosolino, who won the 200 freestyle and 200 individual medley in Helsinki, came through with exceptional power in Sydney. He took gold in the 200 medley, silver in the 400 freestyle and bronze in the 200 freestyle.
Double Olympic champion Pieter van den Hoogenband enters the fray on Tuesday when he begins the defence of his 100 metres freestyle title.
The 26-year-old Dutchman made his first big splash in the 1999 edition of these championships in Istanbul when he won the 50, 100 and 200 freestyle, 50 butterfly and two relay golds.
The following year in Helsinki, Van den Hoogenband had to settle for silvers in the 50, 100 and 200 freestyle, twice beaten by Alexander Popov and once by Rosolino.
But he saved his best for the Olympics when he took the Russian's 100 freestyle title and beat top Australian Ian Thorpe to win the 200freestyle.
At the 2002 Europeans in Berlin, Van den Hoogenband won the 100 and 200, missing the 100 world record he had set at the Sydney Olympics by a mere 0.02 seconds.
Popov, who regained his world 50 and 100 metres freestyle titles in Barcelona last year, will compete only in the 50 freestyle next weekend and will not clash with his Dutch rival in Madrid.
Popov, double Olympic champion in both 1992 and 1996, will race against Mark Foster, who hopes to advance his bid for late selection for the British Olympic team, having missed the standard time demanded by a wafer-thin 0.05 seconds in April's trials.
Foster, silver medallist between Popov and Van den Hoogenband in the 50 freestyle at last year's world championships, has appealed against his exclusion, saying he was hampered by a shoulder injury at the trials.
Foster is the only high-profile British swimmer competing in Madrid.
Germany have also adopted a low profile, with a small squad of just nine pool swimmers, ahead of their Olympic trials in Berlin in early June.
Franziska van Almsick, who led a world record spree at the 2002 Europeans in her native Berlin, triple freestyle world champion Hannah Stockbauer and world backstroke gold medallists Thomas Rupprath and Antje Buschschulte are among prominent German absentees.
Dutch triple Olympic gold medallist Inge de Bruijn, who has skipped virtually all but the Olympics and world championships in recent years, will also be missing but most European champions will defend their titles.
Five of Europe's eight Olympic champions -- Van den Hoogenband, Klochkova, Rosolino, Hungary's Agnes Kovacs (200 breaststroke) and Romania's Diana Mocanu (100 and 200 backstroke) -- and six of the continent's 12 world champions are competing in Madrid.