A number of studies have shown that systemic 5-HT(6) receptor antagonists can improve learning and memory, but the mechanism for these observations is not known. As striatum normally expresses 5-HT(6) receptors abundantly and is important in consolidating stimulus-response learning, we used targeted gene delivery to further increase the expression of 5-HT(6) receptors in rat striatum and then examined learning. Increased 5-HT(6) expression had no effect on performance in the Morris water maze, a hippocampal-dependent learning paradigm, and did not alter the latency to approach or consume sucrose tablets. However, rats with increased 5-HT(6) expression failed to acquire a reward-based instrumental learning task, a striatum-dependent learning model, during 3 days of successive sessions as compared to sham surgery or GFP-expressing control rats. This behavioral deficit was observed in rats overexpressing 5-HT(6) receptors in the dorsomedial striatum, but not in rats with increased dorsocentral striatal expression. The 5-HT(6) receptor-associated deficit was reversed by administration of a 5-HT(6) antagonist, SB-258585, before each training session. When animals learned the instrumental learning task before gene transfer, increased 5-HT(6) receptor expression had no effect on long-term recall or performance of the task or on extinction of operant responding. Thus, 5-HT(6) receptor activity in rat striatum disrupts acquisition of new instrumental learning but does not impair memory or performance of reward-motivated behavior once established.