The genotoxicity of melamine was evaluated with the combined Pig-a mutation/micronucleus assay, the bacterial reverse mutation assay, and the in vitro cytokinesis-block micronucleus assay (CBMN). Five groups of six- to eight-week-old male Sprague–Dawley (SD) rats were given three daily doses of vehicle control (100% pure sesame oil), melamine (500, 1000, and 2000mg/kg) or positive control (N-ethyl-N-nitrosourea, ENU, 20mg/kg) by oral gavage. Peripheral blood was sampled pre-dose (day −1) and at time points up to day 60. Pig-a mutant frequencies were determined in total red blood cells (RBCs) and reticulocytes (RETs) as RBCCD59− and RETCD59− frequencies, on days −1, 15, 29 and 60, and micronucleus frequencies were measured in RETs on day 4. No significant increases in RBCCD59− or RETCD59− frequencies were observed for the melamine-treated group at any of the time points studied, but the positive control, ENU, induced statistically significant increases compared with the vehicle control. Similar results were obtained in the micronucleus assay. Melamine did not induce statistically significant increases in %MN-RET. In the bacterial reverse mutation assay, melamine was tested from 62.5 to 1000μg/plate in tester strains TA97a, TA98, TA100, TA102, and TA1535, with and without metabolic activation, and no evidence of toxicity or mutagenicity was observed at any dose tested. In the in vitro CBMN assay, in Chinese hamster ovary (CHO) cells, melamine was tested (75, 150, and 300μg/mL) in the presence and absence of S9 mix, and no positive increases in the number of cells containing micronuclei were seen. These results suggest that melamine does not exhibit significant genotoxic potential. These data could be valuable for risk assessment purposes and also for further characterizing the new in vivo Pig-a gene mutation assay.