Fifteen lactating Holstein cows were used in a trial designed to evaluate the effectiveness of intrauterine inoculation (challenge) of Actinomyces pyogenes (A) alone or in combination with Fusobacterium necrophorum (F) and Bacteroides melaninogenicus (B) to induce pyometra. Cows were assigned to one of five groups: A (n = 3), AB (n = 3), AF (n = 3), ABF (n = 3) or C (control, broth medium alone; n = 3). All cows exhibited estrus 12 or 13 d prior to challenge (Day 0=first day of challenge). During the prechallenge period, the reproductive tract of each cow was palpated per rectum and uterine fluid aspirates for culture and uterine biopsies were also obtained. All cows received an intravenous injection of 5,000 IU of human chorionic gonadotropin (hCG; Day 5) and an intrauterine infusion of 40 ml of 0.7% iodine solution (Day 1). Cows were then inoculated on Days 0, 1 and 2 of the experiment. Sequential palpations of the reproductive tracts, samples of uterine fluid for culture and uterine biopsies were performed for a total of 30 d after the first inoculation. A cow was diagnosed as having pyometra when purulent uterine fluid and a corpus luteum were detected by palpation per rectum. The number of cows that developed pyometra in Group A was 2 of 3, in Group AB 3 of 3, in Group AF 3 of 3, in Group ABF 3 of 3 and in Group C 0 of 3. Cows with pyometra did not exhibit estrus. In 7 of 11 cows, pyometra persisted for more than 21 d. In cows that developed pyometra, the same species of bacteria infused into the uterus were usually recovered one or more times during the postchallenge period. When clinical pyometra was diagnosed, histologic endometritis was invariably present. Histologic endometritis and concurrent isolation of A . pyogenes alone or A . pyogenes with gram-negative anaerobic bacteria occurred in 91.7% of samples during the postchallenge period. Regardless of bacterial treatment, gram-negative anaerobic bacteria were frequently isolated with A . pyogenes during this period.