We compared biochemical and molecular methods for the identification of heterozygous carriers of mutations in the cystathionine beta-synthase (CBS) gene. Eleven relatives of seven unrelated patients with homocystinuria due to homozygous CBS deficiency and controls were studied with respect to total homocysteine concentrations before and after methionine loading. In addition, we determined CBS activity in cultured skin fibroblasts and tested for the presence of five known mutations by a PCR-based method in these seven patients, their relatives and controls. The results demonstrate that measurement of homocysteine after methionine loading and assay of CBS enzyme activity in cultured fibroblasts identify most but not all heterozygotes. There was significant correlation between homocysteine concentrations and CBS activities only after methionine loading (r = 0.12, 0.48, 0.48 and 0.50 at 0, 4, 6 and 8 h, respectively). Among the homozygous patients, molecular approaches identified five T833C and two G919A mutations out of 14 independent alleles, confirming the studies of others that these represent the two most prevalent mutations. In addition, we found that three of six heterozygotes with the T833 C allele had post-methionine loading homocysteine levels which overlapped with controls and of the other three, one (as well as an obligate heterozygote who did not carry any of the five mutant alleles tested) had CBS activity comparable to that of controls. These findings demonstrate that genotyping is useful as an adjunctive method for the diagnosis of the heterozygous carrier state of CBS deficiency.