Streptomyces venezuelae S13 produced a pH-indicating sporulation pigment on a glucose-salts-agar medium consisting of glucose, KNO(3), MgSO(4), and Na(2)HPO(4), pH 7. Pigmentation on this medium appeared to be closely associated with sporulation, which normally required 5 to 7 days at 30 C. The pigment was soluble in water as well as in a number of organic solvents. Butanol-extracted pigment exhibited absorption maxima at 430 and 520 nm at pH 3 and 12, respectively. Although many salts of organic acids and amino acids could replace glucose as the sole carbon source in basal salts-agar medium for growth and pigmentation, most sugars that were tested supported good growth but negligible pigmentation. Among the nitrogenous substances tested, KNO(3) was most desirable for pigmentation. The organism did not exhibit any specific requirements for divalent cations with respect to growth and pigmentation. In the absence of MgSO(4), however, glucose-salts-agar prepared by autoclaving all components together failed to support growth. The production of the sporulation pigment on glucose-salts-agar was comparable to that obtained on tomato paste-oatmeal-agar medium. Incorporation of partially purified pigment material into broth medium that did not normally support sporulation induced sporulation, and amino acid-salts-agar medium could induce vegetative mycelia to pigment when transferred from medium that did not support either pigmentation or sporulation.