Although aldehyde dehydrogenase (ALDH) from sheep liver cytosol has a broad specificity, it will not oxidize the aldehyde group of glyoxylic acid which is in fact an inhibitor of the enzyme. The inhibition pattern is non-linear but competitive at high propionaldehyde concentrations (2-20 mM); however, a simple non-competitive pattern is observed at low (less than 100 microM) propionaldehyde concentrations (Ki = 1.6 mM). The esterase activity was unaffected by glyoxylic acid in the absence of NAD+ but a simple competitive inhibition pattern (Ki = 2.5 mM) was observed with respect to 4-nitrophenyl acetate in the presence of NAD+. The data require a two-site model in which ester and aldehyde binding sites are distinct but with a second propionaldehyde molecule, and glyoxylic acid, binding at or near the ester binding site. Consistent with this model is the fact that chloral hydrate was a non-competitive inhibitor of the esterase activity in the presence of NAD+ but a competitive inhibitor in its absence. The enzyme exhibited hysteretic behavior governed by the protonated form of an ionizable group with an apparent pKa of 7.55.