The mRNA for the novel aldehyde dehydrogenase 5 (ALDH5) gene was detected in HuH7 hepatoma cells. The cells also expressed cytosolic aldehyde dehydrogenase (ALDH1) mRNA, but no mitochondrial aldehyde dehydrogenase (ALDH2) mRNA. Extracts of the hepatoma cells contained an enzymatic activity with an isoelectric point similar to that of ALDH1. This enzyme activity was insensitive to inhibition by disulfiram, a potent inhibitor of ALDH1. The enzyme was active with short chain aldehydes (acetaldehyde and propionaldehyde) and NAD+, but not with NADP+, and the activity was higher in the mitochondrial pellet than other cell fractions. These studies demonstrate the expression of ALDH5 mRNA in a human hepatoma and suggest that the gene product is enzymatically active and probably resides in the mitochondria.