The opportunistic pathogen Mycobacterium avium is a significant inhabitant of biofilms in drinking water distribution systems. M. avium expresses on its cell surface serovar-specific glycopeptidolipids (ssGPLs). Studies have implicated the core GPL in biofilm formation by M. avium and by other Mycobacterium species. In order to test this hypothesis in a directed fashion, three model systems were used to examine biofilm formation by mutants of M. avium with transposon insertions into pstAB (also known as nrp and mps). pstAB encodes the nonribosomal peptide synthetase that catalyzes the synthesis of the core GPL. The mutants did not adhere to polyvinyl chloride plates; however, they adhered well to plastic and glass chamber slide surfaces, albeit with different morphologies from the parent strain. In a model that quantified surface adherence under recirculating water, wild-type and pstAB mutant cells accumulated on stainless steel surfaces in equal numbers. Unexpectedly, pstAB mutant cells were >10-fold less abundant in the recirculating-water phase than parent strain cells. These observations show that GPLs are directly or indirectly required for colonization of some, but by no means all, surfaces. Under some conditions, GPLs may play an entirely different role by facilitating the survival or dispersal of nonadherent M. avium cells in circulating water. Such a function could contribute to waterborne M. avium infection.