Selenocysteine Se-conjugates have recently been proposed as potential prodrugs to target pharmacologically active selenol compounds to the kidney. Although rat renal cytosol displayed a high activity of beta-elimination activity toward these substrates, the enzymes involved in this activation pathway as yet have not been identified. In the present study, the possible involvement of cysteine conjugate beta-lyase/glutamine transaminase K (beta-lyase/GTK) in cytosolic activity was investigated. To this end, the enzyme kinetics of 15 differentially substituted selenocysteine Se-conjugates and 11 cysteine S-conjugates was determined using highly purified rat renal beta-lyase/GTK. The results demonstrate that most selenocysteine Se-conjugates are beta-eliminated at a very high activity by purified beta-lyase/GTK, implicating an important role of this protein in the previously reported beta-elimination reactions in rat renal cytosol. As indicated by the rapid consumption of alpha-keto-gamma-methiolbutyric acid, purified beta-lyase/GTK also catalyzed transamination reactions, which appeared to even exceed that of beta-elimination. The corresponding sulfur analogs also showed significant transamination but were beta-eliminated at an extremely low rate. Comparison of the obtained enzyme kinetic data of purified beta-lyase/GTK with previously obtained data from rat renal cytosol showed a poor correlation. By determining the activity profiles of cytosolic fractions applied to anion exchange fast protein liquid chromatography and gel filtration chromatography, the involvement of multiple enzymes in the beta-elimination of selenocysteine Se-conjugates in rat renal cytosol was demonstrated. The identity and characteristics of these alternative selenocysteine conjugate beta-lyases, however, remain to be established.