The submitochondrial localization and identity of enzymes which metabolize cysteine conjugates were investigated. Glutamine transaminase K was purified from rat kidney mitochondrial soluble fraction and was shown to be a cysteine conjugate beta-lyase. The purified mitochondrial enzyme is similar to the cytosolic glutamine transaminase K whose beta-lyase activity with S-(1,2-dichlorovinyl)-L-cysteine (DCVC) is regulated by concurrent transamination (Stevens, J. L., Robbins, J. D., and Byrd, R. A. (1986) J. Biol. Chem. 261, 15529-15537). However, beta-lyase activity in whole mitochondria is largely independent of regulation by cosubstrates for transamination suggesting that factors present in mitochondria are able to support the beta-lyase activity in the absence of added alpha-keto acid. Fractionation of mitochondria results in a loss of the independent beta-lyase activity. However, the majority of the beta-lyase activity can be recovered in the matrix if it is stimulated by the addition of alpha-keto-gamma-methiolbutyrate. The data suggest that the regulation of beta-elimination by the competing transamination pathway is different for each substrate and that multiple beta-lyases may exist in rat kidney. S-(2-Benzothiazolyl)-L-cysteine (BTC) is a poor substrate for purified glutamine transaminase K from mitochondria and cytosol, but BTC is as active as DCVC in crude mitochondrial matrix suggesting that other enzymes may be present. In contrast to DCVC, with BTC as substrate, the beta-lyase activity of the purified enzyme and enzyme(s) in the mitochondrial matrix is largely alpha-keto acid-independent. The existence of multiple enzymes is also supported by the observation that alpha-keto acids which are not substrates for purified glutamine transaminase K from mitochondria and cytosol do stimulate beta-lyase activity in the mitochondrial matrix fraction. Mitoplasts were found to be sensitive to DCVC toxicity consistent with the matrix localization of beta-lyase activity. The possible role in cysteine conjugate toxicity of matrix enzyme regulation by alpha-keto acids is discussed.