A metabolite of homocysteine (Hcy), the thioester Hcy thiolactone, damages proteins by modifying their lysine residues which may underlie Hcy-associated cardiovascular disease in humans. A protein component of high density lipoprotein, Hcy thiolactonase (HTase) hydrolyzes thiolactone to Hcy. Thiolactonase is a product of the polymorphic PON1 gene, also involved in detoxification of organophospates and implicated in cardiovascular disease. Polymorphism in PON1 affects the detoxifying activity of PON1 in a substrate-dependent manner. However, how PON1 polymorphism affects HTase activity is unknown. Here we report a strong association between the thiolactonase activity and PON1 genotype in human populations. High thiolactonase activity was associated with L55 and R192 alleles, more frequent in blacks than in whites. Low thiolactonase activity was associated with M55 and Q192 alleles, more frequent in whites than in blacks. High thiolactonase activity afforded better protection against protein homocysteinylation than low thiolactonase activity. These results suggest that variations in HTase may play a role in Hcy-associated cardiovascular disease.