The objective of this study was to elucidate the role of biofilms as the habitat of aquatic mycobacteria. Investigations were carried out on a biofilm which grew on the inner surface of a silicone tube constantly perfused by water of a distribution system known to be contaminated with Mycobacterium kansasii and M. flavescens. The biofilm yielded 2 x 10(5) cfu/cm2 of M. kansasii and 7 x 10(4) cfu/cm2 of M. flavescens after 10 months of perfusion. Microscopic examination revealed that approximately 1/4 to 1/3 of the biofilm organisms visualized by the Ziehl-Neelsen procedure were acid-fast bacteria, most of which occurred in densely packed microcolonies. These findings indicate that biofilms are an important habitat and site for proliferation of aquatic mycobacteria. Biofilms may be an explanation for the problems of controlling mycobacterial contamination of water distribution systems by means of chemical disinfection.