Dopamine agonists with activity at both dopamine D2 and D3 receptor subtypes stimulate tuberoinfundibular dopamine neurons and inhibit prolactin secretion from the anterior pituitary. The purpose of the present study was to identify the dopamine receptor subtypes mediating these effects using recently developed selective agonists for dopamine D2 (PNU-95,666) and D3 (PD128907) receptors. The activity of tuberoinfundibular dopamine neurons was estimated by measuring either the synthesis (accumulation of 3,4-dihydroxyphenyl-alanine [DOPA] following inhibition of decarboxylase activity) or metabolism (3,4-dihydroxyphenylacetic acid [DOPAC] concentrations) of dopamine in the median eminence, the region of the hypothalamus containing axon terminals of these neurons. In one experiment, the activity of mesolimbic dopamine neurons was also determined by measuring DOPA accumulation in terminals of these neurons in the nucleus accumbens. Activation of dopamine D2 receptors with PNU-95,666 caused dose- and time-related increases in DOPAC concentrations in median eminence which were temporally correlated with decreases in plasma prolactin concentrations. Activation of dopamine D3 receptors with PD128907 decreased DOPA concentrations in the nucleus accumbens, but had no effect on concentrations of DOPAC or DOPA in the median eminence or prolactin in plasma. These results reveal that tuberoinfundibular dopamine neurons are regulated by dopamine D2 rather than D3 receptors, and suggest that the ability of mixed dopamine D2/D3 receptor agonists to increase the activity of these neurons is mediated by an action at dopamine D2 receptors. Furthermore, these results confirm that tuberoinfundibular dopamine neurons are not regulated by inhibitory dopamine D2 or D3 autoreceptors.