BRCA1 and BRCA2 germline mutations account for <5% of breast cancer cases. Less penetrant breast cancer susceptibility genes are likely to exist. Earlier studies have suggested involvement of the HLA region. The HLA region was genotyped with 24 microsatellite markers and markers for two single nucleotide polymorphisms (SNPs) in TNFalpha and TNFbeta, in germline DNA from 956 breast cancer patients and 1271 family-based controls. Association analyses and the haplotype sharing statistic (HSS) were used to search for differences in haplotype sharing between patients and controls. Based on criteria known to influence genetic breast cancer risk, patients were divided into groups of high, moderate and low risk. The HSS revealed a significant difference in mean haplotype sharing between patients and controls for four consecutive markers (D6S2671, TNFa, D6S2672 and MICA), the highest being at D6S2671 (P=0.017). Subgroup analyses showed that moderate-risk patients were responsible for this difference, with the strongest association for D6S2672 (P=0.0009). A single haplotype was more frequent and longer in moderate-risk patients than in controls. The results were confirmed with association analyses. Individuals homozygous for haplotype 110-184 (D6S2672-MICA) were observed in 9.0% of moderate-risk patients and 1.5% of controls [odds ratio (OR)=7.14], while heterozygotes were at a lower risk (OR=1.41), suggesting a recessive effect. No association was observed between the two SNPs in TNFalpha (-308) and TNFbeta (intron 1) and breast cancer risk. The results reveal a potential role of the HLA class III subregion in susceptibility to breast cancer in patients at moderate familial risk.