The activity of aldehyde dehydrogenase (ALDH, EC 1.2.1.3) was measured in different fractions of human blood. Of the recovered activity 99% was detected in the intracellular fraction of the erythrocytes. The results also indicated the presence of ALDH activity in the leukocytes, since an increased activity was obtained after cultivation of the cells in the presence of a mitogen. No activity was detected in platelets, plasma, or erythrocyte membranes. Nonlinear Lineweaver-Burk plots were obtained with acetaldehyde, 3,4-dihydroxyphenylacetaldehyde, and indole-3-acetaldehyde as the substrates. The apparent Km values, calculated from the low and high substrate concentration ranges of the curves, were much lower for 3,4-dihydroxyphenylacetaldehyde and indole-3-acetaldehyde than for acetaldehyde. Disulfiram caused almost complete inhibition of the blood ALDH activity in assays with acetaldehyde as the substrate, whereas 15-30% of the activity remained unaffected in assays with 3,4-dihydroxyphenylacetaldehyde and indole-3-acetaldehyde. Kinetic experiments using the mixed substrate method and isoelectric focusing of a partially purified sample of blood did not reveal the presence of more than one isozyme.