The results reported in the present communication were obtained with an organism, Pseudomonas aureofaciens (8, 9), which provides a biological system especially suited for a study of phenazine biosynthesis. Relatively large quantities (1 gm per liter) of the yellow pigment, phenazine-1-carboxylic acid, are produced by growing cultures of P. aureofaciens. The nutritional requirements for optimal production of phenazine-1-carboxylic acid are defined, and some preliminary experiments designed to elucidate the mechanism of biosynthesis of this compound are described. Washed cell preparations of Pseudomonas aureofaciens synthesize phenazine-1-carboxylic acid in a medium consisting of 0.2 M glycerol and 0.02 M D,L-lysine. Experiments with C¹⁴-labeled substrates indicate that all of the carbon atoms in the phenazine-1-carboxylic acid are derived from glycerol. Anthranilic acid inhibits synthesis of this phenazine compound. Incorporation of label from H³-shikimic acid has been demonstrated. In addition, a decrease in the incorporation of label from glycerol-1,3-C¹⁴ into phenazine-1-carboxylic acid has been demonstrated in the presence of unlabeled shikimic acid.