Diethylnitrosamine (DEN) is a genotoxic carcinogen, but in vivo DNA-damaging activities are not usually evident in hematopoietic cells because the short-lived active metabolite is formed mainly in the liver. DEN therefore represented an interesting case for evaluating the performance characteristics of blood-based endpoints of genotoxicity that have been automated using flow cytometric analysis—frequency of micronucleated reticulocytes and Pig-a mutant phenotype reticulocytes (RETCD59−) and erythrocytes (RBCCD59−). Male Sprague Dawley rats were treated for 28 consecutive days with DEN at levels up to 12.5 mg/kg/day. Serial blood samples were collected and micronucleus frequencies were determined on Days 4 and 29, while RETCD59− and RBCCD59− frequencies were determined on Days 15, 29, and 42. The Pig-a analyses were conducted with an enrichment step based on immunomagnetic column separation to increase the statistical power of the assay. Modest but significant reductions to reticulocyte frequencies demonstrated that bone marrow was exposed to reactive intermediates. Even so, DEN did not affect micronucleus frequencies at any dose level tested. However, RETCD59− frequencies were significantly elevated in the high dose group on Day 29, and RBCCD59− were increased at this same dose level on Days 29 and 42. These results demonstrate that the Pig-a assay is sufficiently sensitive to evaluate chemicals for genotoxic potential, even in the case of a promutagen that has traditionally required direct assessment(s) of liver tissue for detection of DNA-damage. Environ. Mol. Mutagen. 55:400–406, 2014. © 2014 Wiley Periodicals, Inc.