We have mutated Asp-251, Thr-252, and Lys-178 in cytochrome P-450cam and studied their effect on steady-state P-450cam catalysis. The mutation of Asp-251 to Asn, which dramatically slows the reaction rate, affects a pH-dependent step in the reaction cycle. By examining the individual steps in the reaction cycle, we have determined that the effect of the D251N mutation occurs after dioxygen binding. Furthermore, our results suggest that the rate-limiting step of the D251N reaction cycle is the O-O bond scission event and that this residue also plays a crucial role in O-O bond scission in wild-type P-450cam. Based on homology with other P-450 enzymes and previous mutagenesis investigations, this role may be common to other P-450 systems, and we suggest a mechanism that is consistent with the effects of these mutations on enzyme activity.