Activation of the dopamine D 1 -like receptor stimulates acetylcholine (ACh) release in the hippocampus, apparently through the molecularly defined d5 receptor. In the present study, we used a transgenic mouse completely deprived of functional d5 receptor (d5À/À) to confirm the role and elucidate the possible function of the d5 receptor subtype on hippocampal cholinergic neurotransmission. ACh release was measured using in vivo microdialysis in the mouse dorsal hippocampus of 4 months old homozygous (d5À/À), heterozygous (d5 þ /À), and the wild-type (d5 þ / þ ) littermates. Using the no net flux technique, a significant reduction in basal hippocampal ACh level was found in the d5À/À compared to d5 þ /À and d5 þ / þ mice. Moreover, the administration of SKF 38393, a D 1 -like receptor agonist, systemically (2.0 and 10.0 mg/kg ip), or locally through the dialysis probe (10 and 50 mM), produced a dose-dependent enhancement of ACh release in the d5 þ / þ , a moderate stimulation in the d5 þ /À but had no effect in the d5À/À mice. Quantitative receptor autoradiography revealed significant increases in M 1 -like but not in M 2 -like muscarinic receptor binding sites in the hippocampal formation. These results confirm and extend the role of the d5 receptor in the modulation of hippocampal ACh release and provide evidence for long-term alteration of hippocampal cholinergic neurotransmission resulting from the absence of the d5 receptors including chronically reduced ACh release and change in M 1 -like receptor levels.