Calcium (Ca2+) ionophore A23187 increased the intracellular cAMP content and PRL release in normal rat anterior pituitary cells. Cotreatment with dopamine reduced both control and A23187-stimulated cAMP accumulation and PRL release. The dopamine antagonist spiperone restored the response of cAMP to ionophoric stimulation after pretreatment with dopamine in the greatest concentration used. Penfluridol, a compound with Ca2+-calmodulin-blocking properties, decreased control and A23187-stimulated cAMP content and PRL release. W7, a selective calmodulin-blocking agent, reduced basal cAMP and PRL release, whereas W5, a W7 analog with only 20% of its calmodulin-blocking ability, did not affect cAMP or PRL secretion. These data indicate that the Ca2+-calmodulin and cAMP systems are interrelated in the regulation of PRL secretion. They are also consistent with the hypothesis that the inhibition of PRL release by dopamine occurs after Ca2+ is mobilized and when or before it stimulates adenylate cyclase activity.