Biofilms have been implicated as reservoirs for bacterial contamination of water delivered by dental air-water syringes. A 6-month study was done of bacterial colonization and biofilm formation in plastic water supply lines connected to dental air-water syringes. Changes in biofilm flora were observed by both scanning electron microscopy and bacteriologic culture. By day 7, many rod- and spiral-shaped bacteria had colonized the ridged surface of the luminal wall of the tubing, as revealed by scanning electron microscopy. By day 30, individual microcolonies were embedded in extracellular polymeric material. By day 120, these microcolonies had begun to coalesce, and by day 180 the biofilm had developed into a multilayered, heterogeneous mixture of microcolonies. The mean aerobic plate counts of colony-forming units of planktonic and biofilm populations were, in log10 values, 5.9 +/- 0.54/mL and 4.2 +/- 0.82/cm2, respectively. Early colonizers were predominantly Pseudomonas spp., but included Pasteurella, Moraxella, Ochrobactrum, and Aeromonas spp. Flavobacterium and Acinetobacter spp. were observed later. Many of these organisms are opportunistic pathogens. These results demonstrate the longitudinal dynamics of biofilm formation.