Cholinergic neurons in the nucleus accumbens express delta- and mu-opioid receptors that are thought to inhibit neural activity. Delta- and mu-opioid receptors are divided into delta1- and delta2-opioid receptors and mu1- and mu2-opioid receptors, respectively. We analysed the roles of delta- and mu-opioid receptor subtypes in regulating accumbal acetylcholine efflux of freely moving rats using in vivo microdialysis. Other than naloxonazine, given intraperitoneally, delta- and mu-opioid receptor ligands were administered intracerebrally through the dialysis probe. Doses of these compounds indicate total amount (mol) over an infusion time of 30–60 min. To monitor basal acetylcholine, a low concentration of physostigmine (50 nM) was added to the perfusate. The delta1-opioid receptor agonist DPDPE (3 and 300 pmol) and delta2-opioid receptor agonist deltorphin II (3 and 30 pmol) decreased accumbal acetylcholine in a dose-related manner. DPDPE (300 pmol)- and deltorphin II (3 pmol)-induced reductions in acetylcholine were each inhibited by the delta1-opioid receptor antagonist BNTX (0.3 pmol) and delta2-opioid receptor antagonist naltriben (15 pmol), respectively. The mu-opioid receptor agonists endomorphin-1 and endomorphin-2 (6 and 30 nmol) decreased acetylcholine in a dose-related manner. Endomorphin-1- and endomorphin-2 (30 nmol)-induced reductions in acetylcholine were prevented by the mu-opioid receptor antagonist CTOP (3 nmol). The mu1-opioid receptor antagonist naloxonazine (15 mg/kg ip), which inhibits endomorphin-1 (15 nmol)-induced accumbal dopamine efflux, did not alter endomorphin-1- or endomorphin-2 (30 nmol)-induced reductions in acetylcholine efflux. This study provides in vivo evidence for delta1-, delta2- and mu2-opioid receptors, but not mu1-opioid receptors, that inhibit accumbal cholinergic neural activity.