Two compounds (celecoxib and valdecoxib) from the diarylheterocycle class of cyclooxygenase inhibitors were radiolabeled and used to characterize their binding to cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), several single-point variants of COX-2 (Val523IIe, Tyr355Ala, Arg120Ala, Arg120Gln, Arg120Asn) and one triple-point variant of COX-2 [Val523IIe, Arg513His, Val434IIe (IHI)]. We demonstrate highly specific and saturable binding of these inhibitors to COX-2. Under the same assay conditions, little or no specific binding to COX-1 could be detected. The affinity of [3H]celecoxib for COX-2 (KD = 2.3 nM) was similar to the affinity of [3H]valdecoxib (KD = 3.2 nM). The binding to COX-2 seems to be both rapid and slowly reversible with association rates of 5.8 × 106/M/min and 4.5 × 106/M/min and dissociation rates of 14 × 10-3/min (t1/2 = 50 min) and 7.0 × 10-3/min (t1/2 = 98 min) for [3H]celecoxib and [3H]valdecoxib, respectively. These association rates increased (4- to 11-fold) when the charged arginine residue located at the entrance to the main hydrophobic channel was mutated to smaller uncharged amino acids (Arg120Ala, Arg120Gln, and Arg120Asn). Mutation of residues located within the active site of COX-2 that define a 'side pocket' (Tyr355Ala, Val523IIe, IHI) of the main channel had a greater effect on the dissociation rate than the association rate. These mutations, which modified the shape of and access to the 'side pocket', affected the binding affinity of [3H]valdecoxib more than that of [3H]celecoxib. These binding studies provide direct insight into the properties and binding constants of celecoxib and valdecoxib to COX-2.