In a recent U.S. Geological Survey/U.S. Environmental Protection Agency study assessing >700 organic compounds in 38 streams, in vitro assays indicated generally low estrogen, androgen, and glucocorticoid receptor activities, but identified 13 surface waters with 17β estradiol equivalent (E2Eq) activities greater than the 1 ng/L level of concern for feminization of male fish. Among the 36 samples assayed for mutagenicity in the Salmonella bioassay (reported here), 25% were considered mutagenic (statistically significant slope and at least a two-fold increase in revertants/plate). Endocrine and mutagenic activities of the water samples were well correlated with each other and with the total number and cumulative concentrations of detected chemical contaminants. To test the predictive utility of knowledgebase-leveraging approaches, site-specific predicted chemical-gene (pCGA) and predicted analogous pathway-linked (pPLA) association networks identified in the Comparative Toxicogenomics Database were compared with observed endocrine/mutagenic bioactivities. We evaluated pCGA/pPLA patterns among sites by cluster analysis and principal component analysis and grouped the pPLA into broad mode-of-action classes. Measured E2Eq and mutagenic activities correlated well with predicted pathways. The pPLA analysis also revealed correlations with signaling, metabolic, and regulatory groups, suggesting that other effects pathways may be associated with chemical contaminants in these waters and indicating the need for broader bioassay coverage to assess potential adverse impacts.