Multiple passage of Legionella pneumophila on either supplemented Mueller-Hinton or charcoal yeast extract agar by the conventional batch passing technique results in loss of virulence. In this study L. pneumophila virulence was maintained after multiple passage on buffered charcoal yeast extract (BCYE alpha) agar by a single-colony transfer technique. Virulence was determined by assessing the growth of L. pneumophila for thioglycolate-induced susceptible A/J mouse macrophages in vitro and infectivity for susceptible A/J mice in vivo. Passage of the virulent L. pneumophila 30 times on BCYE alpha agar by the single-colony transfer procedure still resulted in virulence, as compared with the nonpassaged parental bacteria, both in vitro and in vivo. Lethality for susceptible A/J mice by systemic infection was comparable for the 30th colony-passaged bacteria and the parental L. pneumophila. These results show that the L. pneumophila phenotype associated with intracellular growth in macrophages or infectivity for susceptible mice is stable following passage by the single-colony transfer procedure on BCYE alpha agar.