The specific binding of [125I]insulin to rat adipocytes was reduced in the absence of extracellular calcium. The addition of calcium to the calcium-free medium during incubation restored [125I]insulin binding towards normal. The specific binding of insulin was significantly increased with calcium concentrations as low as 0.5 mM and maximal binding occurred with 5 mM calcium. Scatchard analysis of the data suggests two major binding sites, one a high-affinity low-capacity site (Kd, 1.5 X 10(10) M-1) and the other a lower-affinity high-capacity site (Kd, 4.7 X 10(9) M-1). There was a 50% decrease in the number of high-affinity sites in absence of extracellular calcium. The dissociation curve of receptor-bound insulin was nonlinear both in the absence and presence of extracellular calcium suggesting receptor heterogeneity. The dissociation rate of receptor-bound insulin was greater when insulin was bound in the absence of extracellular calcium than in its presence. These results indicate that extracellular calcium, by increasing the number of high-affinity receptor sites, can alter the the ratio of high-affinity to low-affinity receptors for insulin in adipocytes.