Ca2+ puffs are brief, localized Ca2+ signals evoked by physiological stimuli that arise from the coordinated opening of a few clustered inositol 1,4,5-trisphosphate receptors (IP3Rs). However, the mechanisms that control the amplitude and termination of Ca2+ puffs are unresolved. To address these issues, we expressed SNAP-tagged IP3R3 in HEK cells without endogenous IP3Rs and used total internal reflection fluorescence microscopy to visualize the subcellular distribution of IP3Rs and the Ca2+ puffs that they evoke. We first confirmed that SNAP-IP3R3 were reliably identified and that they evoked normal Ca2+ puffs after photolysis of a caged analog of IP3. We show that increased IP3R expression caused cells to assemble more IP3R clusters, each of which contained more IP3Rs, but the mean amplitude of Ca2+ puffs (indicative of the number of open IP3Rs) was unaltered. We thus suggest that functional interactions between IP3Rs constrain the number of active IP3Rs within a cluster. Furthermore, Ca2+ puffs evoked by IP3R with reduced affinity for IP3 had undiminished amplitude, but the puffs decayed more quickly. The selective effect of reducing IP3 affinity on the decay times of Ca2+ puffs was not mimicked by exposing normal IP3R to a lower concentration of IP3. We conclude that distinct mechanisms constrain recruitment of IP3Rs during the rising phase of a Ca2+ puff and closure of IP3Rs during the falling phase, and that only the latter is affected by the rate of IP3 dissociation.