Media and an analytical scheme have been developed which allow both a qualitative and quantitative estimation of the formation of pyocyanine, related phenazines, pyorubrin, and a blue and a yellow-green fluorescent pigment by Pseudomonas aeruginosa. Use of the defined pyocyanine medium of Frank and DeMoss with sulfate or various organic sulfur sources allowed formation of pyocyanine, related phenazines, and pyorubrin. When sulfite was the sulfur source with or without iron, P. aeruginosa formed either a yellow-green or a blue fluorescent pigment. Formation of fluorescent pigments of P. aeruginosa is related to the ability of sulfite to act as a specific sulfur source. In an investigation of the role of both added iron and sulfur sources, complex patterns of pigment formation were observed. In addition to the fluorescent pigments, sulfite also supported the formation of slime by P. aeruginosa.