Ethanol administration during the rat brain growth spurt triggers apoptotic neurodegeneration that appears to be mediated by caspase-3 activation. In order to gain more insight on the role of this caspase in ethanol-induced developmental neurotoxicity, we studied its expression and activity under different conditions of ethanol exposure during development. Furthermore, because of the cross-talk between caspase-3 and calpain we extended our study also at this protease. Ethanol was administered by gavage to rat pups as a single-day exposure on postnatal day (PN) 7 or from PN4 to PN10. Cleaved caspase-3 expression peaked in the cerebral cortex 12 h after ethanol treatment and returned to control values at 24 h. An identical pattern was found for caspase-3-like activity, that was increased only with the highest dose of ethanol tested (5 g/kg) and mostly in PN4. Repeated ethanol exposure, at a dose that was previously found to induce microencephaly, did not increase caspase-3 expression and activity although it decreased procaspase-3 expression and released mitochondrial cytochrome c. Repeated ethanol administration also increased calpain activity. These data show that acute and repeated ethanol administration differentially affect caspase-3 and calpain activity, suggesting that calpain activation may play a role in developmental neurotoxicity of ethanol.