From normal rat liver mitochondrial and microsomal fractions, 4 distinct aldehyde dehydrogenase isozymes with millimolar substrate Km values have been purified and characterized. Two isozymes were isolated from mitochondria and 2 from microsomes. A mitochondrial aldehyde dehydrogenase with a substrate Km in the micromolar range was also identified. Subunit molecular weights for all millimolar Km isozymes is 54,000. The mitochondrial and microsomal millimolar Km isozymes are clearly distinguishable from each other by substrate and coenzyme specificity, pH velocity profiles, and thermal stability. By these same properties, the 2 isozymes from each organelle are virtually identical. The 2 mitochondrial isozymes can be distinguished by apparent molecular weight (I, 170,000; II, approximately 250,000), Km for NADP+, effect of inhibitors, and pI. The 2 microsomal isozymes are of the same apparent molecular weight (approximately 250,000), but are distinguishable by their Km values for benzaldehyde and NADP+, response to inhibitors, and pI.