In an effort to clarify the pathogenesis of pyometra, 20 cows with retained fetal membranes and 20 without, but with contemporary calving dates were studied. They were palpated and their uteri were subjected to sample collections for bacteriologic cultural examinations twice weekly for 4 weeks. Blood samples were obtained each day and evaluated for serum progesterone concentration. Three cows without and 3 with retained fetal membranes developed pyometra during the study, resulting in 3 groups designated control (CON), cows with retained fetal membranes (RFM), and cows with pyometra (PYO). Bacterial isolations occurred less frequently in the CON group than in the PYO and RFM groups. Growth patterns of bacteria also varied between groups. Coliform and incidental bacteria disappeared from the uterus of the PYO group by the end of the 3rd week. In contrast, heavy growth of Corynebacterium pyogenes and gram-negative anaerobic bacteria developed during this same period in the PYO group. In cows with pyometra, the significant persistent pathogenic bacteria recovered were C pyogenes and gram-negative anaerobic bacteria, especially Fusobacterium necrophorum and Bacteroides melaninogenicus. Anaerobic bacteria were isolated simultaneously with C pyogenes in most cows of the PYO group, but less often in CON and RFM groups, and highest growth levels were present near the time of ovulation. Clinically, pyometra usually developed about 10 days after observation of concurrent ovulation and high growth levels of C pyogenes and gram-negative anaerobic bacteria. A hypothesis is presented for development of pyometra in the cow.