The expirations of 14CO2 from DL-[1-14C]-, DL-[3-14C]- and L-[U-14C] cysteine used as isotopic tracers were estimated in order to determine the in vivo metabolic distribution of L-cysteine in pyridoxine deficient rats. The expired 14CO2 from L-[U-14C] cysteine was increased by pyridoxine deficiency. The loading of non-physiological dose of L-cysteine resulted in remarkable increase in the expiration of 14CO2 from each tracer in deficient rats as well as in controls. The in vivo metabolic distributions of L-cysteine were calculated from the expired 14CO2 from these isotopic tracers. The in vivo metabolic distribution of L-cysteine calculated showed that the remarkable lesion in taurine pathway occurred in pyridoxine deficient rats, and when non-physiological dose of L-cysteine was loaded the catabolism of L-cysteine of controls was markedly increased in either pyruvate or taurine pathway, whereas the L-cysteine catabolism in deficient rats was increased only in pyruvate but not in taurine pathway. The urinary excretions of 35S-labeled metabolites such as sulfate or taurine were also examined in deficient and control rats.