Abstract Localization of myoplasmic free calcium was measured in fura2-loaded single rat myocardial cells to determine whether the mechanism of norepinephrine desensitization includes redistribution of calcium. Fluorescence intensities at each pixel were quantitated by use of a photon-counting, microchannel plate camera. From these images, values of calcium-dependent fluorescence intensity averages in whole cells, areas of calcium release (as zones of high inracellular calcium concentrations), and ratios of fluorescence intensity in central vs. peripheral sites were determined. Stimulation by 1 nM norepinephrine caused an increase in total free intracellular calcium and an activation of intracellular calcium release sites from subsarcolemmal pools initially and later from centrally located calcium pools. Subsequent addition of 100 nM norepinephrine failed to cause significant intracellular calcium release from centrally located pools. In contrast, forskolin exposure still released high concentrations of calcium from these central pools. These results indicate that pretreatment with even a relatively small concentration of norepinephrine causes markedly decreased subsequent intracellular calcium release from centrally located sarcoplasmic reticulum because of a refractoriness of the link between receptor activation and calcium release.