Children of women who smoked during pregnancy are at increased risk of dependence when smoking is initiated during adolescence. We previously reported that gestational nicotine exposure attenuated dopamine release induced by nicotine delivered during adolescence. In this study, we determined the effects of gestational nicotine exposure on nicotinic cholinergic receptor (nAChR) expression. Timed pregnant rats received nicotine (2 mg/kg/day) or vehicle via mini-osmotic pumps during gestation. Treatments continued in pups via maternal nursing during postnatal days (PN) 2-14 (equivalent to the human in utero third trimester). On PN35, 125I-epibatidine binding to nAChR was measured. The Bmax values (fmol/mg) in prefrontal cortex (PFC), nucleus accumbens (NAcc), substantia nigra (SN) and ventral tegmental area (VTA) were reduced by 26.6% (P<0.05), 32.6% (P<0.01), 23.0% (P<0.01) and 27.6% (P<0.05), respectively. In addition, gender differences were found in vehicle-treated groups; in SN and VTA, females were 79.3% (P<0.005) and 82.9% (P=0.08) of males, respectively. The expression of nAChR subunit mRNAs was measured using real-time RT-PCR on laser-capture microdissected tissues. In adolescent VTA, gestational nicotine exposure reduced (P<0.05) nAChR subunit mRNAs encoding alpha3 (53.0%), alpha4 (23.9%), alpha5 (46.7%) and beta4 (61.4%). In NAcc core, the treatment increased alpha3 mRNA (75.8%). In addition, the number of neurons in VTA was reduced by 15.0% (P<0.001). These studies indicate that gestational exposure to nicotine induces long-lasting changes in nAChR expression that may underlie the vulnerability of adolescents to dependence on nicotine.