Enterococcus faecalis plays an important role as one of the pathogens in polymicrobial infections. We evaluated the efficacy of cefluprenam (CFLP) using a polymicrobial pyometra of a model rat. Rats were infected with a mixed intrauterine inoculation ofE. faecalis plus either Bacteroides fragilis or Prevotella bivia (minimal inhibitory concentrations of CFLP: E. faecalis, 3.13 μg/ml;B. fragilis , 3.13 μg/ml; P. bivia, 3.13 μg/ml). Immediately after inoculating 10 5 cfu/rat of each organism, CFLP (either 40 mg/kg, i.v., q.i.d. for 5 days or 80 mg/kg, i.v., b.i.d. for 5 days) was administered. The intrauterine inflammatory change and bacterial count in the treated group were compared with those in the non-treated control group. CFLP significantly (P < 0.01) decreased the bacterial counts except for B. fragilis in the regimen of 80 mg/kg, b.i.d. However, the regimen of 40 mg/kg, q.i.d. significantly (P < 0.05) reduced the bacterial counts more than did that of 80 mg/kg, b.i.d. CFLP proved to demonstrate a good tissue concentration above 3 μg/g for 1 h. these results suggest that CFLP in a more divided dose is efficacious for the treatment of polymicrobial infections associated with E. faecalis in pyometra.