The aim of this study was to determine the effects of artificial insemination on the localization of antigen-presenting cells expressing MHC class II mRNA in chicken oviducts. Laying hens (35 weeks old) were inseminated with fresh semen or sham-inseminated with saline daily for 3 days. In situ hybridization was performed to detect chicken MHC class II (B-LB21 major gene) mRNA on frozen sections of oviductal infundibulum, uterovaginal junction and vagina by using digoxigenin-labelled PCR probes. Cells expressing MHC class II were observed mainly in the oviductal mucosal stroma and occasionally in the mucosal epithelium. After 24 h, the population of cells expressing MHC class II in the infundibulum was significantly higher in laying hens inseminated with fresh semen than in the control hens sham-inseminated with saline (P < 0.05). However, there was no significant difference in the population of cells expressing MHC class II in the uterovaginal junction and vagina between the artificially inseminated and control hens. These results indicate that anti-sperm immune responses, including the influx of cells expressing MHC class II and enhanced MHC class II mRNA expression, probably occur in the infundibulum after artificial insemination.