Experiments were designed to determine if repeated inseminations of egg yolk diluent containing no semen would induce antibodies against egg yolk and would have an adverse effect on subsequent fertility under routine conditions of artificial insemination. Extender containing antibiotics polymyxin, dihydrostreptomycin, and penicillin and the enzyme beta amylase, with or without hen's egg yolk, was inseminated three to four times into virgin dairy heifers. The heifers subsequently were bred with bovine semen suspended in the extender containing egg yolk. Antibodies against antigens in the egg yolk extenders were not detected by complement fixation and agar gel immuno-diffusion, either in blood serum or in whole and homogenized cervical mucus taken before and after the repeat inseminations. Allergic reactions to the semen extender were not observed in any of the heifers. Calving rate from one service was 63.6% (7/11) for controls, 68.2% (15/22) after prior inseminations with egg yolk extender, and 69.2% (9/13) after prior inseminations with extender minus egg yolk. Under routine conditions of artificial insemination three to four exposures to extenders with or without egg yolk did not affect subsequent fertility adversely.