We present here for the first time a method for determining the rate constants associated with slow binding inhibition of prostaglandin H synthase (PGHS). The rate constants were determined by a method using initial steady-state conditions, which minimize the impact of catalytic autoinactivation of the enzyme. The currently available methods for determining the kinetic constants associated with slow binding enzyme inhibition do not distinguish between rate decreases due to enzyme inhibition or due to autoinactivation of the enzyme. A mathematical model was derived assuming a rapid reversible formation of an initial enzyme-inhibitor complex (EI) followed by a slow reversible formation of a second enzyme-inhibitor complex (EI*). The two enzyme inhibitor complexes are assumed to be in slow equilibrium. This method was used to evaluate the kinetic parameters associated with the binding and selectivity of the nonsteroidal antinflammatory drugs (NSAIDs), flurbiprofen and indomethacin. The KI values associated with the formation of the first reversible complex (EI) for flurbiprofen with PGHS1 and PGHS2 were 0.53 +/- 0. 06 and 0.61 +/- 0.08 microM, respectively; the rate constants for the forward isomerization, k2, into the second reversible complex (EI*) were 0.97 +/- 0.99 and 0.11 +/- 0.01 s-1, respectively, and rates of the reverse isomerization from EI*, k-2, were 0.031 +/- 0.004 and 0.0082 +/- 0.0008 s-1, respectively. Indomethacin was estimated to form the EI complex with the same affinity for both PGHS1 and PGHS2, 10.0 +/- 2.8 microM and 11.2 +/- 2.0 microM, respectively, and dissociate from EI* at approximately the same rate 0.0011 +/- 0.0002 s-1 and 0.0031 +/- 0.0003 s-1, respectively. However, the rate of isomerization into EI* from EI was much greater for PGHS1 than PGHS2, 0.33 +/- 0.08 s-1 as compared with 0.034 +/- 0.004 s-1. These results show that the overall affinity for the inhibition of PGHS1 versus PGHS2 was 30-fold greater for indomethacin (KI* = 0.032 +/- 0.005 and 1.02 +/- 0.08 microM, respectively) and 3-fold greater for flurbiprofen (KI* = 0.017 +/- 0.002 and 0.045 +/- 0.004 microM, respectively). The results also show that for both PGHS1 and PGHS2, flurbiprofen was bound tighter to the initial EI complex than indomethacin; however, the rate of dissociation from EI* was slower for indomethacin than flurbiprofen. The rate of the forward isomerization to EI* is primarily responsible for the selectivity of both NSAIDs for PGHS1. This analysis shows the quantitative importance of the different kinetic parameters upon the overall binding affinity of these NSAIDs and should greatly assist in our understanding of the structural interactions that promote enzyme-inhibitor binding.