Cytotoxic activity assays of Gram-negative, heterotrophic bacteria are often laborious and time consuming. The objective of this study was to develop in situ procedures for testing potential cytotoxic activities of heterotrophic bacteria isolated from drinking water systems. Water samples were passed through 0.45 microns membrane filters which were then placed upon appropriate media incubated. After incubation, each membrane filter was transferred to the surface of Y-1 mouse adrenal cells overlaid with 1% agar. The filters were removed after exposure for 15 min. The Y-1 cells were then incubated at 37 degrees C in 2.5% CO2 for an additional 24 h. The release of putative cytotoxic and cytotonic products from the bacterial colonies was recognized by zones of cellular lysis and injury of Y-1 cells that appeared immediately beneath the membrane. Cytotoxic strains of Aeromonas, Vibrio, Escherichia, and Legionella spp. were readily recognized by this method. About 1% of the bacteria isolated from drinking water also released cytotoxic products. This frequency was dependent upon the primary medium used and the density of bacteria present. The majority of cytotoxic strains isolated from drinking water also expressed protease activity (95%) and haemolytic activity (70%). This in situ membrane filter procedure is a facile method for simultaneously testing many different bacterial colonies.