Nine unrelated Legionella micdadei strains isolated from clinical and environmental samples have been characterized biochemically, serologically using polyclonal and monoclonal antibodies and by macrorestriction analyses using pulsed-field gel electrophoresis. All strains were positive in the Bromocresol purple spot test and grew as blue colonies on dye-containing media. They were positive for catalase, weakly positive for oxidase, and negative for sodium-hippurate hydrolysis, beta-lactamase and gelatinase. None of the strains showed autofluorescence under long-wave ultraviolet light. A panel of six monoclonal antibodies raised against the ATCC strain TATLOCK revealed no significant differences in the surface antigen composition of the L. micdadei strains. None of these monoclonal antibodies reacted with L. maceachernii and L. longbeachae serogroup 2, the only species that cross-react with polyclonal antisera. Each of the nine L. micdadei strains showed individual restriction patterns of the genomic DNA when using both SfiI and NotI restriction enzymes in the pulsed-field gel electrophoresis. Macrorestriction analysis is a valuable tool for studies on the molecular epidemiology of L. micdadei.