We tested the hypothesis that progesterone (P₄) withdrawal is the primary mechanism by which intrauterine bacteria induce preterm labor in mice. CD-1 mice on Day 14.5 of a 19- to 20-day gestation were subjected to one of four treatments: 1) intrauterine injection of sterile medium, 2) intrauterine injection of 10⁶ heat-killed Escherichia coli bacteria, 3) intrauterine injection of 10⁹ heat-killed E. coli, or 4) ovariectomy. Mice were then killed at four time points from 0.75 to 11 h after surgery for serum collection. Separately, animals were pretreated either with s.c. P₄ or with vehicle 2 h before ovariectomy or high-dose bacterial inoculation. Ovariectomy led to a rapid fall in serum P₄ levels of 60% by 1 h and 81% by 8 h compared with levels in controls (P < 0.001). In contrast, intrauterine inoculation with 10⁹ bacteria led to a more modest decline in P₄ of only 28% by 8 h (P = 0.24, which was no different from that of 10⁶ bacteria, an inoculum below the threshold for preterm delivery). Despite significantly lower levels of P₄ in the ovariectomy group, time to delivery was significantly shorter with 10⁹ bacteria intrauterine (24 ± 5.6 h vs. 19 ± 3.6 h, P = 0.03). Pretreatment with 1.5 mg P₄ per mouse prolonged the interval to delivery following both ovariectomy and high-dose bacteria, in association with pharmacologically elevated serum P₄ levels. In contrast, physiologic P₄ supplementation (0.375 mg/mouse) prolonged gestation only in the ovariectomy group. We conclude that withdrawal of endogenous P₄ is not the primary cause of labor following intrauterine bacterial inoculation in mice.