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He was born into a working-class family in Prague in 1936 and studied organic chemistry at the Faculty of Mathematics and Physics of Charles University in Prague. After school, he joined the Institute of Organic Chemistry and Biochemistry of the Czechoslovak Academy of Sciences - from an aspirant he became a researcher and his career progressed through senior positions up to the director of the Institute. He belongs to Stanford's worldwide list of the 2% most cited scientists.

Since 1976, he has collaborated with Erik De Clercq of the Catholic University of Leuven, Belgium. Together they are authors of 15 patents. Thanks to the patents and licenses from the research of Antonín Holý and his team, more than 2 billion crowns from the American pharmaceutical company are still coming into the budget of Czech science. The drugs developed are then used worldwide.