The mechanism of action of prostaglandin E1 (PGE1) on rat uterine horns was analyzed. Contractile responses to PGE1 were not due to either release of known neurotransmitters or an interaction with their receptors. Responses could still be elicited in depolarized preparations. PGE1 contractions were Ca++-dependent; the drug appeared to require and utilize Ca++ from the superficial, loosely bound fraction. Anoxia markedly reduced responses to PGE1. Acetylcholine and PGE1 had the following properties in common: (a) neither was potentiated by elimination of external glucose or by lowering of the external K+ concentration, (b) both were still effective in depolarized horns, and (c) pretreatment with adrenaline or 2,4-dinitrophenol and iodoacetic acid reduced responses to both. It is concluded that PGE1 acts on a specific receptor. The receptor–drug interaction may or may not result in subsequent membrane depolarization. Contraction results from an action on the superficial loosely bound calcium fraction, and the processes leading up to this Ca++ release are markedly oxygen-dependent.