Using an in vitro microsuperfusion procedure, the release of newly synthesized [ 3 H]-acetylcholine (ACh), evoked by N-methyl-d-aspartate (NMDA) receptor stimulation, was investigated in striosome-enriched areas and matrix of the rat striatum. The role of μ-opioid receptors, activated by endogenously released enkephalin, on the NMDA-evoked release of ACh was studied using the selective μ-opioid receptor antagonist, β-funaltrexamine. Experiments were performed 2 (morning) or 8 (afternoon) h after light onset, in either the presence or absence (α-methyl-p-tyrosine, an inhibitor of dopamine synthesis) of dopaminergic transmission. As expected, based on the presence of μ-opioid receptors in striosomes, β-funaltrexamine (0.1 nM, 10 nM and 1 μM) enhanced the NMDA (1 mM+10 μM d-serine)-evoked release of ACh in striosome-enriched areas but not in the matrix. Interestingly, these responses were significantly more pronounced in afternoon than in morning experiments. In the presence of α-methyl-p-tyrosine, the NMDA-evoked release of ACh was increased with similar amplitude in morning and afternoon experiments. However, in this condition (without dopamine transmission), the facilitatory effects of β-funaltrexamine on the NMDA-evoked release of ACh were suppressed totally in the morning and only partially in the afternoon. The selective μ-opiate agonist, [d-Ala 2 ,N-Me-Phe 4 ,Gly 5 -ol]-enkephalin (1 μM, coapplied with NMDA), was without effect on the NMDA-evoked release of ACh but abolished both dopamine-dependent (morning) and dopamine-independent (afternoon) responses of β-funaltrexamine (10 nM and 1 μM).Therefore, in the limbic territory of the striatum enriched in striosomes, the μ-opioid-inhibitory regulation of ACh release follows diurnal rhythms. While dopamine is required for this regulation in the morning and the afternoon, an additional dopamine-independent process is present only in the afternoon.