Ten different agar media were tested for the in vitro growth of Mycobacterium genavense in primary cultures and in subcultures from BACTEC vials. These agar media were based on Middlebrook 7H9, 7H10 and 7H11, and supplemented with additives: mycobactin J, yeast extract, charcoal, or defibrinated sheep blood. Some media were acidified with phosphoric acid to a final pH of 6.2 +/- 0.2. Fourteen M. genavense strains from nude mouse organs as well as one decontaminated clinical specimen (from a bird) were tested. The optimal medium for primary cultures of M. genavense was Middlebrook 7H11 acidified to pH 6.2 +/- 0.2 and supplemented with charcoal and sheep blood: on this medium, all strains produced colonies within 6-12 weeks of incubation in numbers approaching the number of bacilli inoculated. It was also the only medium to support the growth of the decontaminated clinical specimen. Added blood and charcoal appeared not as essential for subcultures as for primary cultures. Three media supported the growth of all strains within 1 month incubation: they were acidified, and were supplemented with yeast extract or pancreatic digest of casein, and with either blood or charcoal.