The hydrolysis of p-nitrophenyl acetate (p-NPA) is catalyzed by many proteins. We have observed that oxyhemoglobin A also exhibits esterase activity with a rate intermediate between that of bovine albumin and carbonic anhydrase. Kinetic studies of this reaction revealed that the rate of hydrolysis of p-NPA in the presence of oxy Hb S was approximately 2 times slower than that of oxy Hb A. There is general agreement that the catalytic effect of peptides and proteins on the hydrolysis of p-NPA is mediated by histidines. Oxy Hb Deer Lodge (His beta 2 leads to Arg) hydrolyzes p-NPA at a rate approaching that of oxy Hb S. Oxy Hb F, in turn, exhibits a rate indistinguishable from that of oxy Hb A. The effect of 2,3-diphosphoglyceric acid on the reaction is consistent with the participation of His beta 2 in this catalytic effect. The pH dependence of the reaction and studies with free amino acids also lend support to the involvement of a histidine residue. These results point to subtle conformational differences between oxy Hb S and oxy Hb A in solution, probably involving His beta 2 and/or its microenvironment. The catalytic hydrolysis of p-NPA can be considered a useful probe of conformational states of macromolecules.