Cytochrome P450 1B1 (CYP1B1) is active in the metabolism of estrogens to reactive catechols and of different procarcinogens. Several studies have investigated the relationship between genetic polymorphisms of CYP1B1 and breast cancer risk, however, with inconsistent results. We investigated such an association in postmenopausal Swedish women, with special emphasis on long-term menopausal hormone users, in a large population-based case-control study. We genotyped 1521 cases and 1498 controls for the CYP1B1 single nucleotide polymorphisms (SNPs) m2, m3 and m4 and reconstructed haplotypes. The frequencies of CYP1B1*1, CYP1B1*2, CYP1B1*3 and CYP1B1*4 alleles among controls were estimated to be 0.087, 0.293, 0.444 and 0.175, respectively. It thus appeared that very few haplotypes contained combinations of SNPs at two or three loci and that single SNP genotype data effectively represented haplotypes. Odds ratios (OR) and 95% confidence intervals (CI) were calculated from logistic regression models. We found no overall association between any CYP1B1 genotype and breast cancer risk. The data indicated, however, that women who had used menopausal hormones for 4 years or longer, and carried the CYP1B1*3/*3 genotype may be at increased risk of breast cancer, OR 2.0 (95% CI 1.1-3.5), compared with long-term users without this genotype. We explored the effect of CYP1B1 genotype on breast cancer risk in subgroups defined by body mass index, family history, smoking and catechol-O-methyl transferase genotype, but found no convincing evidence for interaction. In summary, our results strongly indicate that the studied CYP1B1 gene polymorphisms do not influence breast cancer risk overall but may modify the risk after long-term menopausal hormone use.