Although cervical cancer is a common female cancer, little attention has been given to genetic susceptibility factors. The present case-control study was undertaken to examine MTHFR polymorphism as a potential molecular marker of cervical intraepithelial neoplasia (CIN) susceptibility and to relate the findings to smoking, HPV infection, ethnicity, parity and oral contraceptive use, which are known risk factors for cervical cancer. A base change from C to T at the nucleotide position 677 of the MTHFR gene results in substitution of valine (GTC) for alanine (GCC). The homozygous normal (Ala/Ala), homozygous mutant (Val/Val), and heterozygous mutant (Ala/Val) genotypes for the MTHFR gene were determined in cervical tissues of 64 cases of CIN lesions and 31 controls. The genotype frequencies of both Val/Val (17%) and Ala/Val (56%) were significantly higher in subjects with CIN lesions compared to controls with Val/Val (10%) and Ala/Val (39%), (trend p = 0.03). The results suggested a significantly increased CIN risk with an alanine to valine substitution at amino acid 223 of MTHFR with an odds ratio of 2.9 (95% confidence interval: 1.2-7.9, p = 0.02). Age, ethnicity, smoking and oral contraceptive use were weakly and nonsignificantly associated with CIN risk. HPV infection was associated with a statistically nonsignificant threefold increase in CIN risk. Parity and MTHFR genotype displayed a strong interaction. Neither nulliparous women with MTHFR polymorphism nor parous women without the polymorphism were at higher risk than women who did not have children and were MTHFR homozygous normal (the reference category). Women with mutant MTHFR genotype who had children, however, showed a significantly higher risk of CIN, with an odds ratio of 23 (95% confidence interval: 2.3-225) as compared to the reference category. No other factors displayed such a strong pattern of interaction. Since MTHFR polymorphism and pregnancy increases folate requirements and can impair folate status, this association could reflect an inadequate response of mutant MTHFR genotype carriers to the increased demand for folate imposed by pregnancy. Tissue folate deficiency, in turn, could increase the risk of CIN in the affected women.