Ethanol inhibited the proliferation of NPCs. We treated two concentrations (10 mM and 50 mM) of ethanol to rat primary NPCs culture for 1 or 3 days. Cell viability (A) and BrdU incorporation (B) was examined as described in methods. (A) MTT analysis. Ethanol did not induce cellular toxicity against NPCs. (B) Both on day 1 and 3, BrdU incorporation was inhibited by ethanol treatment in a concentration-dependent manner. (C) To investigate inhibitory effect of ethanol on cell proliferation, immunocytochemistry against pH3 or PCNA was performed on day 3. The number of pH3-positive cells as well as PCNA positive cells was reduced by ethanol treatment. (D) FACS analysis of cell cycle. FACS analysis was performed as described in methods 4 hr after ethanol treatment on NPCs culture. Ethanol treatment decreased cells in G2/M phase as compared with control. Values are expressed as the mean ± S.E.M. **, *** p < 0.01 and < 0.001 vs. control (n = 5 for A, B and C. n = 3 for D).