The novel benzoxazolophenanthridine antibiotic, jadomycin B, is produced by Streptomyces venezuelae ISP5230 following a 42 degrees C heat shock or exposure to ethanol. To further characterize these unusual culture conditions, studies were carried out using different media, varying nutrient content and concentrations, initial pH, and time of application of heat or ethanol stress. Highest titers of jadomycin B accumulated 48 h after S. venezuelae ISP5230 was inoculated into a D-galactose-L-isoleucine production medium (pH 7.5) which was supplemented with ethanol (6%, v/v) between 6 and 13 h. Cultures supplemented with ethanol later than 17 h post inoculation into the production medium produced little or no jadomycin B. Among other heat-shock inducing treatments examined, infection with phage SV1 was associated with increased jadomycin B production. Although jadomycin B titers showed little change with variations in the concentration of phosphate in the production medium, the nature of the nitrogen source was found to be important. Different colored pigments, presumed to be jadomycin B analogs, were formed when other amino acids replaced L-isoleucine in the medium as the sole nitrogen source. Increased jadomycin B titers accompanied increased L-isoleucine and D-galactose concentrations in the production medium.