The purpose of this study was to examine the role of the prodynorphin gene in alcohol sensitivity, preference and vulnerability to alcohol consumption. Handling-induced convulsion (HIC) associated to alcohol, alcohol-induced loss of righting reflex (LORR), hypothermic effects in response to acute ethanol challenge, blood ethanol levels (BELs), conditioned place preference, voluntary ethanol consumption and preference, tyrosine hydroxylase (TH), dopamine transporter (DAT) and proenkephalin (PENK) gene expression, and µ-, δ- and κ-opioid agonist-stimulated [S35]- guanosine 5′-triphosphate-binding autoradiography were studied in prodynorphin knockout (PDYN KO) and wild-type (WT) mice. There were no differences in HIC, LORR or the decrease in body temperature in response to acute ethanol challenge between PDYN KO and WT mice. PDYN KO mice presented higher BEL, higher ethanol-conditioned place preference and more ethanol consumption and preference in a two-bottle choice paradigm than WT mice. These findings were associated with lower TH and higher DAT gene expression in the ventral tegmental area and substantia nigra, and with lower PENK gene expression in the caudate–putamen (CPu), accumbens core (AcbC) and accumbens shell (AcbSh) in PDYN KO. The functional activity of the µ-opioid receptor was lower in the CPu, AcbC, AcbSh and cingulate cortex (Cg) of PDYN KO mice. In contrast, δ- and κ-opioid receptor-binding autoradiographies were increased in the CPu and Cg (δ), and in the CPu, AcbC and Cg (κ) of PDYN KO. These results suggest that deletion of the PDYN gene increased vulnerability for ethanol consumption by altering, at least in part, PENK, TH and DAT gene expression, and µ-, δ- and κ-opioid receptor functional activity in brain areas closely related to ethanol reinforcement.