Changes in cytosolic calcium concentration ([Ca2+]i) in response to extracellular calcium and epinephrine were monitored in individual rat adipocytes by both photon counting and digital imaging techniques utilizing the intracellular fluorescent calcium probes Fura-2 and Indo-1. Adipocytes containing Fura-2 were attached to coverslips and shown to be as hormonally responsive to insulin as adipocytes in suspension [3.5 ± 0.8 (n = 5) νs. 4.2 ± 0.6 (n = 8)-fold increase in glucose oxidation over basal in response to 0.7 nM insulin]. Basal [Ca2+]i in single rat adipocytes was found to be 128 ± 6 nM (n = 100). The addition of either extracellular calcium or epinephrine elicited transient, concentration-dependent increases in [Ca2+]i. Although the characteristics of calcium-and epinephrine-induced calcium transients are generally similar, the peak [Ca2+]i increase over basal is higher in response to calcium vs. epinephrine [37 and 64% (1 and 27 epinephrine), νs. 132 and 236% (2 and 4 mM calcium)]. All the cells tested responded to calcium but only 67% responded to epinephrine. Both α-and β-adrenergic agonists were able to increase [Ca2+]i. The epinephrine-induced [Ca2+]i transients appear to be dependent upon extracellular calcium. Neither cholera nor pertussis toxin treatments altered basal [Ca2+]i. However, after treatment of adipocytes with either pertussis or cholera toxin, epinephrine stimulated oscillations in [Ca2+]i. Digital imaging revealed that adipocytes demonstrate a high degree of intracellular spatial heterogeneity and intercellular variability in the magnitude of response to both calcium and epinephrine. These studies demonstrate the feasibility of using single rat adipocytes to monitor intracellular free calcium, using both photon counting and digital imaging. © 1992 by The Endocrine Society.