Samples from Walnut Creek, upstream of the Troy Wastewater Treatment Plant (TWWTP), and the influents to and effluents from the TWWTP were assayed for mutagenicity using the Salmonella typhimurium fluctuation test. Samples were prepared with metabolic activation (channel catfish S9 and rat S9 enzymes) and without using TA100 and TA98 strains of Salmonella. Results indicated that catfish S9 enzymes (FS9) were more capable of activating base-pair substitution mutagens in upstream samples than rat S9 enzymes (RS9). For influent samples, RS9 activated higher levels of base-pair and frameshift mutagens than FS9. The comparison of changes from influent to effluent samples showed a significant reduction in base-pair and no change in frameshift mutagens with FS9; conversely, no change in base-pair and a significant reduction in frameshift mutagens with RS9 were found. For direct-acting compounds (without enzymatic activation), a significant increase in frameshift mutations was found in effluent compared to influent, while no significant change was seen in base-pair substitutions. These results indicate that Walnut Creek contains both mutagenic and promutagenic compounds, and influents to TWWTF exhibit mutagenicity that may be refractory to or created by treatment processes. The generally higher mutagenicity ratios following RS9 activation vs. FS9, suggest that current toxicity studies in fish species and water quality requirements may be inadequate to assess the hazards of water resources that receive municipal wastewater treatment discharges and that may be habitat to both fish and mammalian wildlife and may eventually become sources for human exposures.