3-Oxo-Delta 5-steroid isomerase (Delta 5-3-ketosteroid isomerase, KSI; EC 5.3.3.1) catalyzes the conversion of a variety of beta, gamma-unsaturated 3-oxosteroids to their corresponding alpha, beta-unsaturated isomers at rates that approach the diffusion limit for specific substrates. The reaction proceeds through a dienolate intermediate, with two amino acid residues (Asp-38 and Tyr-14) known to be involved in catalysis. When the complete three-dimensional structure of KSI was determined recently by NMR methods, an additional polar residue (Asp-99) was found in the active site and this group was shown to be important for catalytic activity. In this work, we examine the properties of several mutant KSIs to determine the nature of catalysis by Asp-99 of KSI. The electrophoretic mobilities of wild-type (WT) KSI and several mutants (D99A, D99N, D38N, and D38N/D99A) on native gels were determined at pH values ranging from 6.0 to 8.5. The results demonstrate that the pKa of Asp-99 is >8.5 in wild-type KSI. The pH-rate profiles for the D99A, D99N, and D38H/D99A mutants of KSI were also determined. For all three mutants, kcat and kcat/KM do not decrease at high pH, in contrast to those for WT and D38H, which lose activity above pH 9 and 8, respectively. Mutation of Asp-99 to Asn decreases kcat for the substrate 5-androstene-3,17-dione by 27-fold and kcat/Km by 23-fold, substantially less than the loss of activity (3000-fold in kcat and 2200-fold in kcat/Km) observed when Asp-99 is mutated to Ala, consistent with a hydrogen bonding role for Asp-99. Taken together, these results provide evidence that Asp-99 participates in catalysis in its protonated form, with a pKa of >9 in WT and approximately 8.5 in the D38H mutant. Asp-99 likely donates a hydrogen bond to O-3 of the steroid, helping to stabilize the transition state(s) of the KSI-catalyzed reaction.