Depending on their interaction with intracellular proteins, G proteincoupled receptors (GPCR) often display different affinities for agonists at 37degreesC. Determining the affinity at that temperature is often difficult in intact cells as most GPCRs are internalized after activation. When sequestration of the B-2 bradykinin receptor (B2R) was inhibited by either 0.5 M sucrose or phenylarsine oxide (PAO), a shift in the affinity was detected when the incubation temperature was raised from 4degreesC to 37degreesC or lowered from 37degreesC to 4degreesC. In contrast, binding of the antagonist {[}H-3]NPC 17731 was temperatureindependent. B2R mutants displayed different affinity shifts allowing conclusions on the role of the involved amino acids. By inhibiting receptor sequestration it was possible to determine also dissociation of {[}H-3]BK and of {[}H-3]NPC 17731 from intact cells at 37degreesC. Surprisingly, both dissociation rates were markedly enhanced by the addition of unlabeled ligand, most likely via prevention of reassociation of dissociated {[}H-3]ligand. This suggests that dissociated {[}H-3]ligand cannot move freely away from the receptor. In summary, our data demonstrate that inhibition of receptor internalization either by PAO or sucrose provides an excellent method to study receptor function and the effects of mutations in intact cells.