Uptake of calcium by brush-border membrane vesicles from rat small intestine is composed of saturable and nonsaturable components. We studied regulation of calcium uptake using the divalent cations strontium, a foreign ion that mimics calcium biologically and magnesium, a physiologically important ion that resembles calcium physically, although not biologically. Strontium present outside the vesicle inhibited saturable calcium uptake competitively, consistent with binding to the transporter at the same site as calcium. Strontium inside the vesicle accelerated saturable calcium uptake from the outside (countertransport), also consistent with binding to the same transporter site as calcium. Thus the calcium transporter shows functional characteristics of a mobile carrier. In the uptake medium (extracellular), magnesium was a noncompetitive inhibitor of saturable calcium transport, consistent with a regulatory role in calcium uptake by binding to the transporter at a locus other than that for calcium. Magnesium at 1 mM concentration inside the vesicle had no effect on saturable calcium uptake and a high concentrations functioned as a week uncompetitive inhibitor. Thus intracellular magnesium appears to have no major role in regulating saturable calcium uptake at the brush border of the enterocyte.