ABSTRACT Cells of the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 supplemented with micromolar concentrations of l -[ 14 C]arginine took up, concentrated, and catabolized this amino acid. Metabolism of l -[ 14 C]arginine generated a set of labeled amino acids that included argininosuccinate, citrulline, glutamate, glutamine, ornithine, and proline. Production of [ 14 C]ornithine preceded that of [ 14 C]citrulline, and the patterns of labeled amino acids were similar in cells incubated with l -[ 14 C]ornithine, suggesting that the reaction of arginase, rendering ornithine and urea, is the main initial step in arginine catabolism. Ornithine followed two metabolic pathways: (i) conversion into citrulline, catalyzed by ornithine carbamoyltransferase, and then, with incorporation of aspartate, conversion into argininosuccinate, in a sort of urea cycle, and (ii) a sort of arginase pathway rendering glutamate (and glutamine) via Δ 1 pyrroline-5-carboxylate and proline. Consistently with the proposed metabolic scheme (i) an argF (ornithine carbamoyltransferase) insertional mutant was impaired in the production of [ 14 C]citrulline from [ 14 C]arginine; (ii) a proC (Δ 1 pyrroline-5-carboxylate reductase) insertional mutant was impaired in the production of [ 14 C]proline, [ 14 C]glutamate, and [ 14 C]glutamine from [ 14 C]arginine or [ 14 C]ornithine; and (iii) a putA (proline oxidase) insertional mutant did not produce [ 14 C]glutamate from l -[ 14 C]arginine, l -[ 14 C]ornithine, or l -[ 14 C]proline. Mutation of two open reading frames ( sll0228 and sll1077 ) putatively encoding proteins homologous to arginase indicated, however, that none of these proteins was responsible for the arginase activity detected in this cyanobacterium, and mutation of argD ( N -acetylornithine aminotransferase) suggested that this transaminase is not important in the production of Δ 1 pyrroline-5-carboxylate from ornithine. The metabolic pathways proposed to explain [ 14 C]arginine catabolism also provide a rationale for understanding how nitrogen is made available to the cell after mobilization of cyanophycin [multi- l -arginyl-poly( l -aspartic acid)], a reserve material unique to cyanobacteria.