Secretion of prolactin is tonically inhibited by hypothalamic release of dopamine into the hypophysial portal system. However, the role that changes in dopamine secretion play in altering prolactin secretion after physiologic stimuli is still unknown. The present study was designed to investigate changes in dopamine release into stalk blood during suckling-induced release of prolactin. An increase in prolactin secretion was induced in urethane-anesthetized, lactating rats by a 15-min electrical stimulation of an isolated mammary nerve trunk. This procedure induced a rapid increase in prolactin secretion, and a 7-fold increase in prolactin concentrations was observed within 20–30 min after stimulation began. In unstimulated control rats, prolactin levels remained at baseline values during the period of observation. Then, we measured the effect of this stimulus on the concentration of dopamine in hypophysial stalk plasma. Dopamine concentrations in hypophysial stalk plasma, collected at 15-min intervals before, during and after mammary nerve stimulation, decreased significantly by 20% during stimulation, returned to prestimulation values, and then increased significantly by 20% at 45–60 min. In control rats, no changes in dopamine concentrations were observed. These results demonstrate that a simple, inverse relationship between dopamine secretion and prolactin secretion does not exist during suckling. However, the observed decrease in dopamine secretion during mammary nerve stimulation may be an integral part of a complex mechanism, including other hypothalamic hormones, that lead to the release of prolactin.