The mammalian kidney is composed of numerous cell populations associated with the interstitial spaces, vasculature and various portions of the nephron. Not surprisingly, mammalian kidneys also exhibit an array of drug metabolizing activities, including the pyridine nucleotide-linked aldehyde dehydrogenase(s) (ALDH). To define the ALDH activity in segment 2 of the proximal tubule, a portion of the nephron which frequently shows drug-induced pathology, proximal tubules were isolated by purely mechanical methods from female rabbits. Isopycnic centrifugation of tubule-derived postnuclear supernates in linear sucrose gradients resulted in propionaldehyde (5 mM)-driven ALDH activity being distributed in a manner consistent with both a mitochondrial and cytosolic localization. Mitochondrial and cystosolic fractions yielded biphasic reciprocal plots when propionaldehyde was used as substrate. Km values of 282 microM and 4 mM were obtained from the mitochondrial enzymes, whereas the cytosolic enzymes gave KmS of 132 microM and 2.4 mM. The apparent Vmax values (nanomoles of NADH produced per minute per milligram of protein) are 16.3 and 37.8 for the mitochondrial enzymes and 23.5 and 19.0 for the cytosolic enzymes. Thus, the S2 proximal tubule cells contain ALDH activities at levels and subcellular sites comparable to those found in the liver. Because the proximal tubules constitute approximately 40% of the renal cortical mass, the high levels of ALDH activity observed in these cells may protect other cortical cells and more distal nephron components by detoxifying potentially cytotoxic aldehydes.