Microinjections of the cholinergic receptor agonist nicotine and the cholinesterase inhibitor neostigmine were made into the ventral tegmental area (VTA) of urethane-anesthetized rats, and dopamine (DA) efflux in the nucleus accumbens was measured using in vivo chronoamperometry. Dose-dependent increases in the chronoamperometric signals corresponding to increased DA efflux were observed in the nucleus accumbens of normal intact rats after cholinergic stimulation of the VTA. The source of the cholinergic input to the VTA was investigated by making excitotoxic lesions in either the laterodorsal tegmental nucleus (LDTg) or the pedunculopontine tegmental nucleus (PPTg). Compared with sham-operated control animals, which showed the same response as intact, nonlesioned rats, ibotenate lesions of the LDTg attenuated the stimulatory effects of intra-VTA neostigmine on DA efflux in the nucleus accumbens. In contrast, rats with ibotenate lesions of the PPTg showed normal nucleus accumbens DA eflux after intra-VTA injections of neostigmine. Such lesions in the PPTg attenuate DA efflux in the caudate-putamen stimulated by injections of neostigmine into the substantia nigra pars compacta (SNc). The present data show that cholinergic neurons in the LDTg, but not the PPTg, regulate the activity of DA-containing neurons in the VTA, which complements previous data showing that cholinergic neurons in the PPTg regulate DA-containing neurons in the SNc.