To clarify the regulation of prostaglandin F2 alpha (PGF2 alpha) production in vivo in mice during pregnancy and the estrous cycle, we injected [3H]PGF2 alpha i.v. into female mice and determined the structures of three urinary metabolites by gas chromatography-mass spectrometry. The compounds were 5,7,11-trihydroxy-tetranor-prosta-9-enoic acid (FUM-I), 5,7,11-trihydroxy-tetranor-prostanoic acid (FUM-II), and 5,7-dihydroxy-11-keto-tetranor-prostanoic acid (FUM-III). The major metabolite, FUM-III, increased four-fold at the term of pregnancy and transiently during diestrus of the estrous cycle. Consistent with the increase in FUM-III, immunoblot analysis with anti-bovine PGF synthase antiserum demonstrated that a 36-kDa protein band corresponding to PGF synthase increased in the uterus at late pregnancy and during diestrus. These results suggest that PGF2 alpha production may increase at term and change during the estrous cycle and is associated with an increase in uterine PGF synthase concentrations.