Hemoglobin adducts of the carcinogenic tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) release 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) upon mild base or acid hydrolysis. HPB has been detected in hydrolysates of hemoglobin from smokers and snuff dippers and has been proposed as a dosimeter of exposure to and metabolic activation of NNK in people exposed to tobacco products. In this study, labeling experiments were carried out with [18O]NaOH that provide strong evidence that the globin adduct that releases HPB upon hydrolysis is a carboxylic ester. Globin was isolated from rats treated with [5-3H]NNK. This globin was reacted with NaCNBH3, followed by hydrolysis at room temperature with 0.2 N NaOH. Analysis of the products demonstrated the presence of 4-hydroxy-1-(3-pyridyl)l-butanol, but not HPB. These results demonstrate that the adduct in globin has a free carbonyl group and is not a Schiff base. This sequence of reactions was then carried out with [18O]NaOH under conditions that were shown to result in incorporation of 18O if nucleophilic displacement at C-4 had occurred. Analysis by GC-MS of the 4-hydroxy-1-(3-pyridyl)-1-butanol formed in this experiment demonstrated that there was no incorporation of 18O. These results are consistent only with the hydrolysis of an ester by a B(AC)2 mechanism. Therefore, the adduct releasing HPB upon mild base hydrolysis must be a 4-(3-pyridyl)-4-oxobutyl ester of aspartate, glutamate, or a terminal carboxylate. Further support for this conclusion was obtained by investigating the chemistry of a model ester, α-methyl-β-4-(3-pyridyl)-4-oxobutyl-N-carbobenzyloxy-L- aspartate. The hydrolysis properties of this compound were similar to those of the HPB-releasing hemoglobin adduct.