Workplace exposures to mixtures of nickel compounds have been associated with excess respiratory cancer risk. Animal studies with individual nickel compounds indicate that not all nickel substances have the same potency or potential to induce tumors. The bioavailability of nickel ions at critical cellular sites seems to be important to determine the potential of a substance to induce tumors in animals, but much less is understood about the exact nature (genotoxic or non-genotoxic) of the nickel effects. Within many regulatory frameworks (e.g., European Union), substances are classified for mutagenicity based on the available data and this classification will often influence the mode of action assigned to carcinogenic substances and the way in which risk assessment will be conducted. The objective of this study was to evaluate the ability of nickel sulfate hexahydrate to induce micronuclei in polychromatic erythrocytes (PCEs) in rat bone marrow. This study was conducted according to OECD and EU protocol guidelines. In the dose range-finding assays, the maximum tolerated dose was estimated to be 500 mg/kg/day. The doses used in the micronucleus assay were 125, 250, and 500 mg/kg/day. At least 2000 PCEs per animal were analyzed for micronuclei in PCEs. Cytotoxicity was assessed by scoring a minimum of 500 consecutive total polychromatic (PCE) and normochromatic (NCE) erythrocytes (PCE/NCE ratio). Nickel sulfate hexahydrate did not induce statistically significant increases in micronucleated PCEs at any dose examined. The negative results in the present study contribute significantly to the weight of evidence evaluation of the mutagenicity (chromosomal level) of nickel substances. These results are consistent with a non-genotoxic mode of action for soluble nickel that could explain the enhancement of cancer risk seen among refinery workers with mixed exposures and its lack of carcinogenicity in animal studies with single exposures.