In bitches, the onset of pyometra, an infection of the uterus, characteristically occurs in the first half of the diestrous stage in the estrous cycle, in which the blood concentration of progesterone peaks and that of estradiol-17beta is lowest. To investigate the immunological mechanisms governing stage-specific onset of pyometra, peripheral blood mononuclear cells (PBMNCs) were collected from beagle bitches during different stages of the estrous cycle and examined using various immunological assays. When we examined the proliferative response of PBMNCs to PYO-252, that is a clone of Escherichia coli isolated from the uterus of a dog afflicted with pyometra, the response of PBMNCs significantly decreased in the first half (day 10) of diestrus, but increased in proestrus/estrus. No significant differences were observed in the responses to concanavaline A between stages of the cycle. Throughout the estrous cycle, canine PBMNCs did not respond to lipopolysaccharide derived from E. coli. The response of PBMNCs collected in anestrus to PYO-252 was significantly enhanced upon the addition of estradiol-17beta to the culture. In contrast, these responses were significantly suppressed in the presence of progesterone. Progesterone progenitor or metabolite molecules, which have a low affinity for the progesterone receptor, did not affect proliferative responses. Expression of gamma interferon (IFNgamma) in response to PYO-252 was also significantly enhanced by estradiol-17beta, but suppressed by progesterone. This evidence suggests that in the first half of the diestrous stage, suppressed activity of cellular immunity results from increasing progesterone concentration and minimal estrogen release. This marked decrease of immune resistance allows the expansion of E. coli, which enter the uterine cavity through the loosened cervical canal during estrus, leading to pyometra onset.