A comparison of the actions of ethanol and acetaldehyde on glucose production from various precursors by isolated rate liver cells was made to evaluate the role of acetaldehyde in the effects of ethanol. Both ethanol and acetaldehyde stimulated glucose production from pyruvate, inhibited glucose production from glycerol, xylitol, and sorbitol, and both had no effect with fructose. Ethanol, but not acetaldehyde, inhibited glucose production from lactate, dihydroxyacetone and alanine. The inhibitory effects of ethanol and acetaldehyde were prevented by methylene blue, an aritificial electron acceptor. The similarities in the actions of ethanol and acetaldehyde on glucose production from some substrates, e.g. pyruvate, glycerol, xylitol and sorbitol suggest that the metabolism of acetaldehyde may contribute to the effects of ethanol on gluconeogenesis from these substrates. Differences in the actions of ethanol and acetaldehyde on glucose production from other substrates reflect the different compartments, cytosol for ethanol, mitochondrial for acetaldehyde, in which these compounds are metabolized in the liver.