Only a small percentage of the heterotrophic bacteria encountered in water distribution systems are identifiable because many of these organisms fail to grow on the conventional media used for biochemical characterization. Organisms that would not subculture from the same standard plate count agar used for initial isolation were successfully subcultured on a low-nutrient medium, R3A. These cultures were then inoculated to a modified O/F base medium containing specific substrates. This, combined with a lower incubation temperature (30 C), increased the enzymatic activity of many of the organisms. These reactions established a groundwork for tentative taxonomy.