Water hardness is well known to affect the toxicity of some metals; however, reports on the influence of hardness during incubation or acclimation on later toxicity to metals have been conflicting. We incubated rainbow trout (Oncorhynchus mykiss) near the confluence of two streams, one with soft water and one with very-soft water (average incubation hardnesses of about 21 and 11 mg/L as CaCO₃, respectively). After developing to the swim-up stage, the fish were exposed for 96-h to a mixture of cadmium (Cd) and zinc (Zn) in water with a hardness of 27 mg/L as CaCO₃. The fish incubated in the higher hardness water were about two times more resistant than the fish incubated in the extremely soft water. This difference was similar or greater than the difference that would have been predicted by criteria hardness equations had the fish been tested in the different acclimation waters. We think it is plausible that the energy demands for fish to maintain homeostasis in the lower hardness water make the fish more sensitive to metals that inhibit ionoregulation such as Cd and Zn. We suggest that if important decisions were to be based upon test results, assumptions of adequate hardness acclimation should be carefully considered and short acclimation periods avoided. If practical, incubating rainbow trout in the control waters to be tested may reduce uncertainties in the possible influences of differing rearing water hardness on the test results.