Acute toxicity tests using Daphnia magna or D. pulex have been employed for many years to determine the toxicity of effluents and toxic substances. However, the many factors that may affect the results of such tests have received little attention. Sequential, repetitive tests were conducted with the reference toxicants sodium dodecyl sulphate (SDS), sodium pentachlorophenate (PCP), and cadmium (Cd) to determine the single laboratory precision of acute 48-h toxicity tests using D. magna and D. pulex, and the effects of feeding on toxicity. The sensitivity of Daphnia to pH shock was also examined. Control survival was 90% or better in 84% of the tests, and 80% or better in 89% of the tests. Survival of controls in tests where the organisms were fed was slightly higher than when they were not fed. The precision of the method, expressed as the relative standard deviation (percent coefficient of variation or CV) of the 48-h LC⁵⁰, for each reference toxicant, SDS, PCP, and Cd, was 43.8, 35.7, and 20.9% for D. pulex and 28.9, 10.4, and 72.4% for D. magna, respectively. No statistically significant differences (P = 0.05) were observed in 48-h LC⁵⁰ values obtained from paired tests with fed and unfed D. pulex exposed to PCP or Cd, or with D. magna exposed to SDS. However, the 48-h LC⁵⁰ values were significantly different for fed and unfed D. magna exposed to PCP or Cd. D. pulex and D. magna were equally sensitive to SDS and PCP, but D. magna was significantly more sensitive to Cd than D. pulex, even though D. magna was tested in water of much greater hardness. Sudden changes in pH of not more than two pH units in the range of pH 5 to 9 resulted in no mortality over a six-day period.