Fresh bovine semen was contaminated with five strains of Campylobacter fetus at 10(5) to 10(6) organisms per ml of semen. The contaminated semen was processed to contain 500 units of polymyxin B, 500 units of penicillin G, and 1000 micrograms of streptomycin sulfate per ml, incubated at 35 degrees C for 10 min, and added then to whole milk extender with like units of antibiotics for further processing. Both a low and high initial sperm count requiring extension rates 1:7 and 1:39 were studied. Specimens for bacterial viability were processed for culture prior to adding bacteria, 100 min after initial antibiotic treatment, prior to freezing, and after thawed semen was centrifuged and washed to remove extender and antibiotic residue. All strains of Campylobacter fetus were not viable 6 h postantibiotic treatment prior to freezing. All nonantibiotic treated controls were viable after thawing and washing. Removal of antibiotics from thawed semen by centrifugation and washing indicated that antibiotics had bactericidal rather than only bacteriostatic activities against Campylobacter fetus.