L-Kynurenine 3-hydroxylase [EC 1.14.1.2] was partially purified from the mitochondrial outer membrane fraction of Saccharomyces carlsbergensis by Sephadex G-200 gel chromatography, and the effects of leucine and its related compounds on the enzyme were investigate. Alpha-Keto acid derivatives of the the three branched chain amino acids were found inhibitory to the partially purified kynurenine 3-hydroxylase, but branched chain amino acids were without effect. Alpha-Ketoisocaproate (KIC), a keto acid analogue of L-leucine, inhibited kynurenine 3-hydroxylase noncompetitively with apparent Ki values of 4.2 and 8.3 mM for kynurenine and NADPH respectively. Alpha-Ketoglutarate and pyruvate were mixed-type inhibitors of the enzyme. KIC production by S. carlsbergensis grown in medium containing no leucine was negligible, while that in leucine incorporated into cells. From the results, it was proposed that KIC produced from leucine lowered synthesis of NAD from tryptophan by inhibiting L-kynurenine 3-hydroxylase, a possible rate-limiting enzyme in the tryptophan-NAD pathway in Saccharomyces carlsbergensis.