The mycobacterial flora of 321 water samples was explored to evaluate the role of this part of the environment as a possible source of human mycobacterial disease. The samples included natural waters, waters treated to make them suitable for drinking, and waters in contact with animals. Water from the city aquarium contained the greatest abundance of mycobacteria, with an average of 3.5 strains per sample. The highest yield of positive cultures came from samples in contact with zoo animals and with fish. The majority of the isolated strains were slowly growing mycobacteria; 80 were Mycobacterium gordonae, and 34 of thse belong to a new serotype. Forty-seven cultures were members of the M. avian-intracellulare-scrofulaceum complex, of which 11 were typable by agglutination. From this study and from the work of others, it is concluded that water may be contaminated with potentially pathogenic mycobacteria and thus may serve as a source of human disease.