An in vitro cytotoxicity methodology, initially developed for use with mammalian cells to evaluate the relative acute toxicities of chemical agents, has been adapted for aquatic ecotoxicity studies by using cultured fish cells as the bioindicator system. This methodology, termed the neutral red assay, was applied to evaluating the comparative in vitro cytotoxicities of a series of polychlorinated biphenyl mixtures and substituted toluenes to bluegill sunfish BF-2 cells in culture. Although comparatively small differences were noted in the in vitro cytotoxicities of the polychlorinated biphenyl mixtures, their differential potency to the BF-2 cells was Aroclor 1248 ≃ 1016 ≃ 1254 ≃ 1242 > 1221 ≃ 1232 > 1260 > 1262. For the toluene series, the sequence of comparative potency was α,α,2,6-tetrachlorotoluene > 2,3-dinitrotoluene > 2,4-dichlorotoluene, 6-chloro-3-hydroxytoluene > 2,6-dinitrotoluene, 2,4-dinitrotoluene, o-, m-, and p-chlorotoluene >>> toluene. The in vitro cytotoxicities of the chlorinated toluenes, but not of the nitro-containing toluenes, were correlated with their log octanol/water partition coefficients. These data are in accord with published studies on the in vivo acute toxicities (LC₅₀ assays) to fish of chloro- and nitro-containing toluenes. The greater in vitro cytotoxicity of 2,3-dinitrotoluene than of the two other dinitrotoluene analogs was also in agreement with published in vivo acute toxicity studies with freshwater fish. This present study, in conjunction with previous in vitro cytotoxicity assays with BF-2 cells, has demonstrated the potential usefulness of the neutral red assay for predicting the in vivo response of fish to acute exposures of chemicals and for establishing structure-activity relationships among groups of related chemicals.