Ca<sup>2+</sup> transients (1–2 µ M) evoked by serotonin (5-HT) in cultured A<sub>7</sub>r<sub>5</sub> cells were studied using fura-2 and digital imaging microscopy. Fura-2 was introduced into cells either by incubation with its acetoxymethyl ester analogue fura-2/AM or by transient ATP-induced permeabilization of the sarcolemma such that the free fura-2 entered the cell directly. The distribution of cytoplasmic Ca<sup>2+</sup> in unstimulated cells loaded by the former method was heterogeneous, reflecting, in part, separate pools of Ca<sup>2+</sup> in the cytosol and sarcoplasmic reticulum (SR). In contrast, the distribution of Ca<sup>2+</sup> was uniform in cells loaded with fura-2 by transient permeabilization; this reflected the restriction of fura-2 to the cytosol. Average Ca<sup>2+</sup> in these cells was substantially lower than that in fura-2/AM-loaded cells, because SR Ca<sup>2+</sup> influences the fura-2 signal from fura-2/AM-loaded cells, but not from cells loaded with free fura-2. The differences in the Ca<sup>2+</sup> distribution measured by the two loading methods were also evident during the course of 5-HT-evoked Ca<sup>2+</sup> transients. Spatial and temporal resolution of the rising phase of 5-HT-evoked Ca<sup>2+</sup> transients in fura-2/AM-loaded cells revealed that the onset of the Ca<sup>2+</sup> transients was first manifested as small regions of elevated Ca<sup>2+</sup> that subsequently expanded until peak apparent intracellular Ca<sup>2+</sup> levels were present in virtually all of the nonnuclear regions of the cells. The rate of rise of Ca<sup>2+</sup> varied in different cell regions with the nucleus responding the slowest.