Elevated plasma levels of low density cholesterol and their major apolipoprotein (apo B) are associated with an increased risk of coronary artery disease (CAD). We have examined allele frequencies of restriction fragment length polymorphisms (RFLP) of the apo B gene in 111 male Caucasians with premature CAD (mean age 49 +/- 7 years) and in 122 elderly Caucasian males (mean age, 73 +/- 5 years), free of clinical cardiovascular disease. The rare allele (R1) of the EcoR1 RFLP in exon 29, resulting in an amino acid change (Glu----Lys4154) was seen more frequently in CAD than in controls (0.270 vs 0.207, P less than 0.05). The R1 RFLP and the MspI insertion polymorphisms (MI) within the 3' hypervariable region (HVR) were observed together in 87% and are likely in linkage disequilibrium. The MI RFLP were slightly more frequent in CAD than control (0.239 vs. 0.211, P = 0.08). A second MspI RFLP in exon 26 results in an amino acid change (Arg----Glu3611); the rare allele M2 was seen more frequently in patients than in controls (0.150 vs. 0.057, P less than 0.005). No significant differences in allele frequencies were observed for the Xba1 RFLP in exon 26 (0.500 vs. 0.529, P = ns) or for the PvuII RFLP near the 5' end (P2) (0.105 vs. 0.088, P = ns). No statistically significant differences in lipid, lipoprotein cholesterol or apolipoproteins A-I and B were observed in patients or in controls. Two of the RFLPs examined (R1 and M2) result in changes in amino acid sequence and their allele frequencies are increased in CAD cases when compared with controls. Genetic variability within the apo B gene may thus contribute to cardiovascular risk. The physiological effects of individual mutations within apo B remain to be determined. It is unlikely, however that the single site polymorphisms examined in this study, will impart further information about CAD risk than conventional lipid parameters.