Nanotoxicity studies are greatly needed to advance nanomedical technologies into clinical practice. We assessed the toxic effects of a single intravenous exposure to commercially available gold nanoparticles (GNPs) in mice and rats. Fifteen-nm GNPs were purchased and independently characterized. Animals were exposed to either 1,000 mg GNPs/kg body weight (GNP group) or phosphate-buffered saline. Subsets of animals were euthanized and samples collected at 1, 7, 14, 21, and 28 days postexposure. Independent characterization demonstrated that the physicochemical properties of the purchased GNPs were in good agreement with the information provided by the supplier. Mice exposed to GNPs developed granulomas in the liver and transiently increased serum levels of the pro-inflammatory cytokine interleukin-18. No such alterations were found in rats. While there was no fatality in mice post-GNP exposure, a number of the rats died within hours of GNP administration. Differences in GNP biodistribution and excretion were also detected between the two species, with rats having a higher relative accumulation of GNPs in spleen and greater fecal excretion. In conclusion, GNPs have the ability to incite a robust macrophage response in mice, and there are important species-specific differences in their biodistribution, excretion, and potential for toxicity.