The effects of independently altering chloride, calcium, and dissolved organic carbon (DOC) on the toxicity of silver (presented as AgNO3) were compared between rainbow trout (Oncorhynchus mykiss) and fathead minnows (Pimephales promelas). The 96-h median lethal concentration (LC50) toxicity tests for both species were performed under the same conditions, within the same containers. In addition, the effect of altering [Cl−] on silver-induced perturbations to body Na+ influx and gill silver load was studied. Toxicity tests were conducted in synthetic soft water (50 μM Na+, 50 μM Cl−, 50 μM Ca2+, 0.3 mg DOC/L). The [Cl−], [Ca2+], and [DOC] were adjusted by the addition of NaCl, CaNO3, or humic acid, respectively. On the basis of total silver, increasing [Cl−] over a range of 50 μM to 1,500 μM resulted in a 4.3-fold increase in the 96-h LC50 values (decrease in toxicity) for rainbow trout, but did not significantly affect the 96-h LC50 values for fathead minnows. Increasing water [Ca2+] (from 50 to 2,000 μM) had only a small influence (1.5-fold increase) on the 96-h LC50 values in either species. However, increasing DOC levels (from 0.3 to 5.8 mg DOC/L) significantly increased the 96-h LC50 values (2.7- to 4.1-fold increases) in both species. If the 96-h LC50 values are calculated on the basis of ionic silver, Ag+ (utilizing the aquatic geochemical computer program MINEQL+ ), then, in the case of rainbow trout, toxicity correlates to Ag+. However, this correlation does not exist for fathead minnows. Increasing [Cl− did not affect the degree of perturbation of Na+ influx during acute exposure (first 4 h) to 8 μg Ag/L in either species, nor did it affect the whole-body silver uptake rates, but it did reduce the gill silver load. These results demonstrate that differences exist in the way in which water chemistry ameliorates silver toxicity between rainbow trout and fathead minnows.