1. The ability of oxytocin and prostaglandin F2 alpha (PGF) to induce contraction in guinea‐pig myometrium in calcium‐free solution was studied in an attempt to assess the extent to which intracellular calcium stores could be released by these two agonists. Both longitudinal and circular muscle layers were studied separately and the effects of gestational age were also examined. 2. In longitudinal strips, the responses to oxytocin and PGF in the absence of external calcium decreased progressively throughout gestation. Responses of circular strips to both agonists were unchanged throughout pregnancy, until day 64, when no response to PGF could be elicited. 3. Pre‐treatment with high potassium (and normal calcium) increased the responses to the agonists in calcium‐free medium while pre‐treatment with beta‐adrenoceptor agonists had no effect on responses to oxytocin or PGF. 4. Responses to both agonists decreased with time in calcium‐free solution suggesting a loss of calcium from stores with a half‐time of 3 min. The rate of the decline in the responses was the same in both muscle layers and did not change with gestational age. 5. In the presence of lanthanum contractions evoked by oxytocin, but not PGF, were augmented 2‐3‐fold. This potentiation of the response to oxytocin occurred in both muscle layers and throughout gestation. 6. Each agonist evoked only one response in calcium‐free solution containing EGTA. The response to PGF in longitudinal strips following a challenge with oxytocin was reduced, compared with the response to PGF when applied first while the response to oxytocin in these strips was unchanged following exposure to PGF. In circular strips neither oxytocin‐ nor PGF‐induced contractions were altered following prior exposure to the other agonist. 7. It is concluded that oxytocin and PGF operate via two distinct mechanisms to release intracellularly stored calcium in both longitudinal and circular components of the guinea‐pig myometrium and a hypothesis to explain the results is presented.