We studied the calcium dependency of the stimulation of prostaglandin synthesis which occurs when perfusing strips of guinea pig Taenia coli with potassium-free media. Stimulation was rapidly reversed by removal of extracellular Ca from the bathing solution. The Ca ionophore A23187 markedly stimulated prostaglandin E2 synthesis, an effect that is dependent on the presence of extracellular Ca. Prostaglandin E2 production in strips in potassium-deficient media was also sensitive to increases in extracellular Ca, and was augmented at concentrations of 7-15 mM. In strips which had been incubated with [3H]arachidonic acid, exposure to potassium-free media caused an increased release of [3H]arachidonic acid and [3H]prostaglandin E2. Release of these labeled compounds with the strips in potassium-free media was further augmented by increasing extracellular [Ca2+] from 2.5 to 10 mM. Treatment with the Ca antagonist agent verapamil did not influence activation of prostaglandin synthesis by potassium-deficient media. The presence of Mn2+ of Ba2+ had similar effects on prostaglandin synthesis, although they had opposite effects on mechanical activity. We conclude that a plasma membrane associated Ca pool is involved in activation of phospholipid metabolism which results in release of esterified arachidonic acid and subsequent prostaglandin synthesis. This Ca pool is in rapid equilibrium with extracellular Ca, is not influenced by cytoplasmic Ca, and is not related to Ca involved in Ca gating in the surface membrane. These data also indicate dissociation between processes involved in muscle contraction and activation of prostaglandin synthesis.