Crude papaya latex (CPL) and its proteinases, papain (PPN) and chymopapain (CPN), are strong uterine contractants. The current study was carried out to examine possible mechanisms of the uterine stimulating activity of the proteinases. Inactivation of the enzymatic activity of papaya proteinases reversibly abolished their uterine-stimulating effect, suggesting that enzymatic activity of the proteinases is a prerequisite for their oxytocic activity. Moreover, removal of Ca²⁺ from the uterine bathing medium reversibly abolished the uterine-stimulating effect of the proteinases. Nifedipine and verapamil (Ca²⁺ channel blockers) significantly and reversibly block CPL-, CPN-, and PPN-induced uterine contractions. Blockade of 5-hydroxytryptamine receptors did not prevent the oxytocic activity of papaya proteinases. However, uterine contractions induced by the proteinases were significantly and reversibly inhibited by meclofenamic acid (a cyclooxygenase and prostaglandin inhibitor). At 0.3 and 1 mg/ml, CPL, CPN, and PPN caused a concentration-dependent increase in prostaglandin F₂ production in cultured rat uterus, but only CPL (1 mg/ml) induced PGF₂ production by the cultured rat uterine tissues was statistically significant. The results of the current study suggest that prostaglandin release by Ca²⁺ mobilization- and proteolysis-dependent activity of papaya latex and its proteinases could play a major role in their oxytocic activity.