Atrazine (2-chloro-4-[ethylamino]-6-[isopropylamino]-1,3,5-triazine) is one of the most commonly used herbicides in North America and is frequently detected in ground and surface waters. This research investigated possible covalent modifications of hemoglobin following in vivo exposures to atrazine in Sprague Dawley (SD) rats and in vitro incubations with diaminochlorotriazine. SD rats were exposed to 0, 10, 30, 100, and 300 (mg atrazine/kg)/day for 3 days via oral gavages, and blood was drawn at 0 h, 24 h, 72 h, 20 days, 1 month, and 2 months for globin analysis. Globin was purified from red blood cells, separated with high-performance liquid chromatography, and analyzed with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). An additional beta globin peak was seen in exposed animals during the HPLC and MALDI-TOF MS analysis with a mass 110 Da greater than the normal beta subunits. Tryptic digests of this beta peak contained a peptide of 1449.9 m/z that corresponded to a modified peptide of amino acids 121-132. Mass spectrometry sequencing of this peptide indicated a 110 Da addition to Cys-125 of the major beta globin chain, which corresponds to a nucleophilic substitution reaction with a diaminochlorotriazine. In vitro incubations of SD globin and diaminochlorotriazine also resulted in a peptide of 1449.6 m/z that was identical in sequence to the modified peptide seen in the in vivo digest, confirming the nucleophilic substitution mechanism of adduct formation. Exposures of SD rats to atrazine results in formation of an adduct that is easily detected and provides an analytical model for detection of triazine adducts in other macromolecules with sulfhydryl functional groups.