Previous studies from our laboratory demonstrated that long-term hypoxia (LTH) altered in vitro contractile responses to oxytocin in full-thickness myometrial strips from pregnant sheep. The present study was designed to determine, first, if the reduced contractile response to oxytocin following LTH is the result of combined effects on longitudinal and circular smooth muscle or if the effect is specific to a single muscle layer and, second, if the reduced contractile response to oxytocin following LTH is caused by changes in oxytocin-receptor protein. Pregnant ewes were maintained at high altitude (3820 m) from Day 30 to Days 137–142 of gestation, when the ewes were killed for collection of myometrial tissue. Tissue was also collected from age-matched, normoxic controls. Longitudinal and circular layers were separated, length-tension curves generated to determine optimal resting tension, and all strips exposed to increasing half-log doses of oxytocin ranging from 10^–12 to 10^–6.5 M. The expression of oxytocin-receptor protein was measured using Western blot analysis. We found that LTH did not affect KCl-induced contraction of either smooth muscle layer, whereas the sensitivity of both myometrial layers to oxytocin was altered. A decreased maximum contractile response of the circular layer to oxytocin was also observed. Additionally, LTH decreased expression of oxytocin-receptor protein in the circular layer and increased levels in the longitudinal layer. Results from the present study indicate that LTH alters contractile responses and oxytocin-receptor protein expression in a layer-specific manner in the pregnant sheep myometrium.