P araoxonase was identified as a genetic risk factor for cardiovascular disease (CVD) in recent studies focusing on a polymorphism affecting position 191. A second polymorphism of the paraoxonase gene affects position 54 and involves a methionine (M allele) to leucine (L allele) change. It was investigated in diabetic patients (n = 408) with and without vascular disease. There were highly significant differences in plasma concentrations and activities of paraoxonase between genotypes defined by the 54 polymorphism: MMAA, MLAA, LLAA; protein, 65.3+/-18.0, 77.9+/-18.0, 93.5+/-26.0 microg/ml; P < 0.0001: activity (phenylacetate), 48.6+/-13.5, 64.1+/-14.5, 68.1+/-13.0 U/ml; P < 0.0001. The 191 variant had little impact on paraoxonase concentrations. Homozygosity for the L allele was an independent risk factor for CVD (odds ratio 1.98 (1.07-3.83); P = 0.031). A linkage disequilibrium (P < 0.0001) was apparent between the mutations giving rise to leucine and arginine at positions 54 and 191, respectively. The study underlines that susceptibility to CVD correlates with high activity paraoxonase alleles. The 54 polymorphism would appear to be of central importance to paraoxonase function by virtue of its association with modulated concentrations. The latter could explain the association between both the 54 and 191 polymorphisms and CVD.