We developed and validated an enzyme immunoassay for immunoreactive pregnanediol-3-glucuronide (iPdG) in feces to monitor reproductive status in desert and Rocky Mountain bighorn sheep (Ovis canadensis). Fecal iPdG concentrations were strongly correlated (r = 0.71) with serum progesterone concentrations in paired fecal and blood samples collected from 34 free-ranging desert bighorn sheep. In bimonthly fecal samples collected from 12 captive ewes, fecal iPdG profiles were similar between desert and Rocky Mountain bighorn sheep and we selected a pregnancy detection cutoff value of iPdG > or = 1.8 ng/mg feces. Fecal iPdG concentrations always exceeded this cutoff value when samples were collected from about day 60 of pregnancy to a few days before parturition, but values < 1.8 ng/mg (false negatives) were common for samples collected during the first 60 days of gestation. Although we tested a small number of known pregnant and non-pregnant ewes, the accuracy of the assay was 100% when two samples, collected 2 wk apart, were evaluated for any given ewe. Based on these data, this direct enzyme immunoassay for fecal progesterone metabolites has promise as a diagnostic tool to monitor hormone excretion and pregnancy in a free-ranging ungulate species.