The building blocks for intracellular Ca2+ signals evoked by inositol 1,4,5-trisphosphate receptors (IP3Rs) are Ca2+ puffs, transient focal increases in Ca2+ concentration that reflect the opening of a small clusters of IP3Rs. We use total internal reflection fluorescence microscopy and automated analyses to detect Ca2+ puffs in human embryonic kidney 293 cells evoked by photolysis of caged-IP3 or activation of endogenous muscarinic receptors with carbachol. Ca2+ puffs evoked by carbachol initiated at an estimated 65 ± 7 sites/cell, and the sites remained immobile for many minutes. Photolysis of caged-IP3 evoked Ca2+ puffs at a similar number of sites (100 ± 35). Increasing the carbachol concentration increased the frequency of Ca2+ puffs without unmasking additional Ca2+ release sites. By measuring responses to sequential challenges with carbachol and photolysis of caged-IP3, we established that the two stimuli evoked Ca2+ puffs at the same sites. We conclude that IP3-evoked Ca2+ puffs initiate at numerous immobile sites, the sites become more likely to fire as the IP3 concentration increases, and there is no evidence that endogenous signalling pathways selectively deliver IP3 to specific sites.