Stable, orange, acriflavine-resistant variants were selected by treatment of a wild-type, red, acriflavine-sensitive strain of Serratia marcescens with acriflavine. Visible, ultraviolet, infrared, and nuclear magnetic resonance spectra of purified pigment from the red strain were identical to those of the pigment from the orange strain, and the orange mutant was not due to a mutation affecting the structure of the pigment, prodigiosin. The color of the red strain was not affected by variations in pH between 5.0 and 8.0, whereas the color of the orange mutant changed from pink to orange over the same pH range. This variation was mimicked by the pH-induced variation in color of prodigiosin purified from either the red, wild-type or the orange, mutant strains. Density-gradient centrifugation of cell fragments after ultrasonic disintegration resulted in characteristic pigmented bands. Biochemical characterization of these pigmented bands showed that they contained pigment and a protein component, but no lipids, polysaccharides, sugars, glucosamine, or phosphates were detected. Further fractionation of these pigmented bands by zone electrophoresis on a sucrose density gradient indicated that some pigment in S. marcescens was specifically attached to protein components.