The objective of this study was to develop a reliable model for the study of the cystic endometrial hyperplasia and pyometra complex (CEH/P) in the bitch. Greyhound bitches (n = 15) were ovariectomised and allocated into three groups (Group 1, n = 5; Group 2, n = 5; Group 3, n = 10, including 5 used from Group 1). Simulated proestrus, estrus and diestrus were induced by treatment with estradiol benzoate and megestrol acetate. The duration of cervical opening during estrus was determined by the intra-vaginal infusion of radio-opaque medium and subsequent radiography of the uterus (Group 1). One milliliter of a culture of Escherichia coli (with five uro-pathogenic virulence factors as identified by PCR: pap, sfa, hlyA, cnf1 and fim) was inoculated intra-vaginally daily throughout the simulated estrus (Group 2). One milliliter of the culture (n = 6) or sterile Luria-Bertani broth (n = 4) was introduced directly into the uterus on simulated diestrus Days 8 or 12 (Group 3). Necropsies were performed 12 and 7-14 days after the inoculation (Groups 2 and 3). The cervix remained open throughout the duration of simulated estrus (5-6 days) in four out of five bitches, and for a shorter duration (3 days of a 6-day estrus period) in one bitch (Group 1). CEH/P was induced by inoculation of bacteria into the uterus (10/10 bitches) but not into the vagina (0/5 bitches), (P = 0.003). A model for the study of CEH/P has been validated.