Water from 34 sites on two temporarily vacant hospital floors was analyzed for the presence of mycobacteria. These sites included 18 cold water taps and 16 hot water taps, including shower heads. A total of 14 sites (41%) demonstrated the presence of Mycobacterium avium as confirmed by biochemical characterization, DNA/rRNA probe analysis, and seroagglutination. Of positive sites, 11 were hot water sources with an average temperature of 55°C and yielding up to 500 colony-forming units per 100 mL. Seven of 11 strains analyzed for glycolipid antigens were identified with the type 4 serovar, the preponderant serovar of M avium in patients with acquired immunodeficiency syndrome from the Boston area. Potable hot water systems, particularly those that generate aerosols, may contain concentrations of M avium greater than those found in cold water systems and could serve as an environmental source for colonization and infection of immunocompromised persons.