To assist Kentucky in refining numeric nutrient criteria in the Pennyroyal Bioregion, the U.S. Geological Survey and the Kentucky Division of Water collected and analyzed water chemistry, turbidity, and biological-community data from 22 streams throughout the Crawford-Mammoth Cave Upland ecoregion (U.S. Environmental Protection Agency Level IV Ecoregion, 71a) within the Pennyroyal Bioregion from September 2007 to May 2008. Statistically significant and ecologically relevant relations among the stressor (total phosphorus, total nitrogen, and turbidity) variables and response (macroinvertebrate-community attributes) variables and the breakpoint values of biological-community attributes and metrics in response to changes in stressor variables were determined. Thirteen of 18 macroinvertebrate attributes were significantly and ecologically correlated (p-value < 0.10) with at least one nutrient measure. Total number of individuals, Ephemeroptera-Plecoptera-Trichoptera richness, and average tolerance value were macroinvertebrate measures that most strongly correlated with the concentrations of nutrients. Comparison of the average macroinvertebrate-breakpoint value for the median concentration of total phosphorus (TP, 0.033 mg/L) and for median concentration of total nitrogen (TN, 1.1 mg/L) to Dodds' trophic classification for TP and TN indicates streams in the Crawford-Mammoth Cave Uplands ecoregion within the Pennyroyal Bioregion would be classified as mesotrophic-eutrophic. The biological breakpoint relations with median concentrations of TP in this study were similar to the U.S. Environmental Protection Agency proposed numeric TP criteria (0.037 mg/L), but were 1.5 times higher than the proposed numeric criteria for concentrations of TN (0.69 mg/L). No sites were impacted adversely using median turbidity values based on a 25 Formazin nephelometric turbidity unit biological threshold. The breakpoints determined in this study, in addition to Dodds' trophic classifications, were used as multiple lines of evidence to show changes in macroinvertebrate community and attributes based on exposure to nutrients.