We report here the first laboratory studies simulating the conditions for natural aerosolization of Mycobacterium intracellulare and M. scrofulaceum and estimate the yields for this pathway of transfer of pathogenic mycobacteria from water to air; M. intracellulare and M. scrofulaceum were both concentrated in droplets ejected from cell suspensions of densities comparable to those found in natural freshwaters (100 to 2,000 colony-forming units per ml). The enrichment factor (defined as the concentration of cells per droplet volume divided by the concentration of cells in the bulk suspension per equivalent volume) for M. intracellulare isolates ranged from 68 to 15,000, with an average of 2,922; for M. scrofulaceum it ranged from 35 to 550, with an average of 177. One factor responsible for the greater aerosolization of M. intracellulare was their aggregation. However, after vortexing, M. intracellulare were still aerosolized more (enrichment factor, 325) than M. scrofulaceum. Increasing salt concentrations enriched the aerosolization of both species, but the number of organisms transferred from water to air did not increase proportionately because the salt decreased the droplet volume. Other waterborne pathogens such as Legionella pneumophila were also enriched and transferred from water to air, indicating that this pathway for possible infection of humans may also be significant for other respiratory diseases.