The National Toxicology Program (NTP) is using the Comet assay to evaluate genotoxic potential, and is investigating the integration of this assay into repeat-dose toxicity studies. To reduce sample-to-sample variability, address logistical concerns associated with evaluating multiple tissues from many animals, and accommodate sample collection at geographically distant testing facilities, tissue samples collected for Comet analysis by the NTP are routinely flash-frozen in liquid nitrogen and stored in a –80°C freezer until evaluation. To compare data obtained from frozen tissues to data from freshly isolated tissues, we conducted a dose-response study in male Sprague Dawley rats. Rats (5 per treatment group) were administered ethyl methanesulfonate (EMS; 0, 25, 50, 100, or 200 mg/kg) by gavage twice at an interval of 21 hr; blood, liver, stomach, and colon tissues were harvested 3 hr after the second treatment. Single-cell preparations from each of the four tissues were put into Hank's balanced salt solution with 10% fresh dimethyl sulfoxide. One aliquot of each tissue preparation was used for immediate analysis, while additional aliquots were flash-frozen in liquid nitrogen and stored in a −80°C freezer for 1 or 8 weeks. One set of 8-week frozen samples was shipped roundtrip via air courier from Research Triangle Park, NC to Rochester, NY prior to analysis. For all four tissues, results from frozen, nontransported samples showed a similar dose-response pattern for EMS-induced genotoxicity. We also demonstrated that for three tissues (blood, liver, stomach), air transport did not alter the sensitivity of the Comet assay for detecting DNA damage. Environ. Mol. Mutagen., 2012. Published 2011 Wiley Periodicals, Inc.