We have studied the 3-mercaptopyruvate pathway (transamination pathway) ofL-cysteine metabolism in rat liver mitochondria.L-Cysteine and other substrates at 10 mM concentration were incubated with mitochondrial fraction at pH 8.4, and sulfate and thiosulfate were determined by ion chromatography. WhenL-cysteine alone was incubated, sulfate formed was 0.7µmol per mitochondria from one g of liver per 60 min. Addition of 2-oxoglutarate and GSH resulted in more than 3-fold increase in sulfate formation, and thiosulfate was formed besides sulfate. The sum (A + 2B) of sulfate (A) and thiosulfate (B) formed was approximately 7-times that withL-cysteine alone. Incubation with 3-mercaptopyruvate resulted in sulfate and thiosulfate formation, and sulfate was formed with thiosulfate. These reactions were stimulated with glutathione. Sulfate formation fromL-cysteinesulfinate and 2-oxoglutarate was not enhanced by glutathione and thiosulfate was not formed. These findings indicate thatL-cysteine was metabolized and sulfate was formed through 3-mercaptopyruvate pathway in mitochondria.