Arginine biosynthesis and its regulation by the presence of different carbon and nitrogen sources in the growth medium of Mycobacterium smegmatis was studied. Replacement of glycerol by glucose and fructose increased the activities of acetylglutamate kinase, acetylornithinase and ornithine transcarbamylase and the enzyme activities of the arginine biosynthetic pathway. The presence of succinate, fumarate, pyruvate or acetate in the growth medium (replacement for citrate) also increased these enzyme activities. However, when glutamate or glutamine was used as nitrogen source in place of asparagine, the enzyme activities decreased. The presence of ornithine or arginine in the growth medium repressed these enzyme activities, though the degree of repression was slight. The phenomenon of repression by arginine and ornithine was confirmed by dialysis experiments. Arginine inhibited the ornithine transcarbamylase activity from cells grown with asparagine as nitrogen source, but activated it when the cells were grown with arginine. Thus, in addition to the weak transcriptional control of arginine biosynthetic enzymes, feedback regulation of ornithine transcarbamylase by arginine also regulated arginine biosynthesis in M. smegmatis grown with asparagine as nitrogen source.