Legionella pneumophila is the causative agent of Legionellosis in man and considered an opportunistic intracellular Gram-negative bacterium that preferentially infects macrophages. The presence of a plasmid in these organisms was determined in cultures of the bacteria grown in vitro. A correlation was observed between the growth of virulent strains of the Legionella in murine macrophages and growth on standard buffered charcoal yeast extract agar supplemented with 0.1% alpha-ketoglutarate (BCYE alpha) agar medium rich in cysteine and widely used for growth of the bacteria in vitro. In contrast, the avirulent isolates of these strains grew well on supplemented Mueller-Hinton (SMH) agar utilized for differentiating virulent from avirulent Legionella. However, one virulent strain of Legionella (the Iowa strain) was found to grow moderately well on the SMH agar. In addition, test strains of Legionella that infect in vitro human monocytes were found to grow moderately well on the BCYE-alpha agar, but did not grow on the SMH agar. Examination of these strains for plasmid DNA expression showed that extra chromosomal DNA-containing molecules were present in the L. pneumophila strains characterized as virulent for in vitro growth in macrophages. However, the avirulent strains that replicated in the human monocytes readily but only poorly in the permissive murine macrophages did not show evidence of similar plasmid DNA expression.