Substance abuse typically begins in adolescence; therefore, the impact of alcohol during this critical time in brain development is of particular importance. Epidemiological data indicate that excessive alcohol consumption is prevalent among adolescents and may have lasting neurobehavioral consequences. Loss of cholinergic input to the forebrain has been demonstrated following fetal alcohol exposure and in adults with Wernicke-Korsakoff syndrome. In the present study, immunohistochemistry for choline acetyltransferase (ChAT) was determined to assess forebrain cholinergic neurons (Ch1–4), and behavioral changes following periadolescent alcohol exposure. Wistar rats were exposed to intermittent ethanol vapor (14 hrs on/10 hrs off/day) for 35 days from PD 22-PD 57 (average blood alcohol concentration (BAC): 163 mg%). Rats were withdrawn from vapor and assessed for locomotor activity, startle response, conflict behavior in the open field, and immobility in the forced swim test, as adults. Rats were then sacrificed at day 71/72 and perfused for histochemical analyses. Ethanol vapor exposed rats displayed: increased locomotor activity 8 hrs after the termination of vapor delivery for that 24 hr period at day 10 and day 20 of alcohol vapor exposure, significant reductions in the amplitude of their responses to prepulse stimuli during the startle paradigm at 24 hrs withdrawal, and at two weeks following withdrawal, less anxiety-like and/or more “disinhibitory” behavior in the open field conflict, and more immobility in the forced swim test. Quantitative analyses of ChAT immunoreactivity revealed a significant reduction in cell counts in the Ch1–2 and Ch3–4 regions of the basal forebrain in ethanol vapor exposed rats. This reduction in cell counts was significantly correlated with less anxiety-like and/or more “disinhibitory” behavior in the open field conflict test. These studies demonstrate that behavioral measures of arousal, affective state, disinhibitory behavior and ChAT+IR, are all significantly impacted by periadolescent ethanol exposure and withdrawal in Wistar rats.