The types and quality of data needed to determine relationships between chronic whole effluent toxicity (WET) test results and in-stream biological condition were evaluated using information collected over a 1.5-y period from 6 different sites across the United States. A data-quality-objectives approach was used that included several proposed measurement quality objectives (MQOs) that specified desired precision, bias, and sensitivity of methods used. The 6 facilities used in this study (4 eastern and 2 western United States) all had design effluent concentrations >60% of the stream flow. In addition to at least quarterly chronic Ceriodaphnia dubia, Pimephales promelas (fathead minnow), and Selenastrum capricornutum (green algae) WET tests, other tests were conducted to address MQOs, including splits, duplicates, and blind positive and negative controls. Macroinvertebrate, fish, and periphyton bioassessments were conducted at multiple locations upstream and downstream of each facility. The test acceptance criteria of the US Environmental Protection Agency (USEPA) were met for most WET tests; however, this study demonstrated the need to incorporate other MQOs (minimum and maximum percent significant difference and performance on blind samples) to ensure accurate interpretation of effluent toxicity. More false positives, higher toxicity, and more “failed” (noncompliant) tests were observed using no-observed-effect concentration (NOEC) as compared to the IC25 endpoint (concentration causing ≥25% decrease in organism response compared to controls). Algae tests often indicated the most effluent toxicity in this study; however, this test was most susceptible to false positives and high interlaboratory variability. Overall, WET test results exhibited few relationships with bioassessment results even when accounting for actual effluent dilution. In general, neither frequency of WET noncompliance nor magnitude of toxicity in tests were significantly related to differences in biological condition upstream and downstream of a discharge. Periphyton assessments were most able to discriminate small changes downstream of the effluent, followed by macroinvertebrates and fish. Although sampling methods were robust, more replicate samples collected upstream and downstream of each facility were needed to increase detection power. In general, macroinvertebrate and periphyton assessments together appeared to be sufficient to address project objectives.