Cystathionine β synthase (CBS) is a key enzyme in homocysteine metabolism. We have examined four apparently non-functional polymorphisms in the CBS gene and have determined their frequency, degree of linkage disequilibrium and association with plasma homocysteine levels. The polymorphisms are a 68 bp insertion in exon 8, C699T in exon 8, C1080T in exon 11 and C1985T in the 3′ untranslated region. 785 individuals participating in the European Atherosclerosis Research Study II (EARSII), from 11 countries across Europe were genotyped for these polymorphisms. The 68bp insertion had the highest frequency in the UK and in the Middle region, with a lower frequency in the Baltic and the South (p=0.01), and the exon 11 polymorphism had the highest frequencies of the rare allele in the Baltic (p<0.05). There was a high degree of linkage disequilibrium between the polymorphisms (p<0.001 overall), except between C699T and the C1985T, with three common haplotypes accounting for nearly 80% of chromosomes. Examination of the association between these polymorphisms and plasma homocysteine levels revealed that the carriers of the rare alleles of the C699T, C1080T and C1985T polymorphisms had lower plasma homocysteine concentrations than those homozygous for the common alleles, although these differences were not statistically significant. The thermolabile valine variant caused by a substitution of a C for a T at nucleotide 677 in the methylenetetrahydrofolate reductase (MTHFR) has previously been shown to have profound effects on plasma levels of homocysteine in this sample, but the homocysteine-raising effect associated with this thermolabile variant was not seen in carriers of the 68 bp insertion, with this interaction being statistically significant (p<0.001). These data demonstrate that variation in the CBS gene as detected with these four polymorphisms, had no statistically significant effect on plasma homocysteine levels in these healthy young men. However, the presence of the 68 bp insertion, which is found in approximately 7.5% of individuals in the populations of Europe sampled, abolishes the raising effect of thermolabile MTHFR Val/Val genotype, and may be of importance in the situation of high homocysteine.