The cell-medium distribution of the nonmetabolized glucose analog, 3-O-methyl-D-glucose was studied in pigeon erythrocytes. The sodium ionophore monensin increased in parallel and in a dose-dependent manner the influx of hexose and of Na+. These effects were independent of external Ca2+ and there was no alteration in 45Ca influx. If, as suggested previously, hexose transport in these cells is modulated by cytoplasmic Ca2+, the stimulatory effect of monensin on hexose transport may be due to increased mitochondrial Ca2+ efflux via Na+-Ca2+ exchange, owing to the elevation of cytoplasmic Na+. Such a mechanism is consistent with the observed failure of monensin to affect 3-O-methyl-D-glucose transport in cells partially depleted of Ca2+. Monensin also depressed cellular ATP levels but the data favour a Ca2+-dependent mechanism of hexose transport regulation rather than a direct effect of metabolic depletion. The inhibitor of specific-mediated hexose transport, cytochalasin B was found to inhibit equally basal and stimulated 3-O-methyl-D-glucose uptake but there was a cytochalasin B-insensitive uptake component in excess of L-glucose uptake. This appears to reflect a greater diffusional permeability of 3-O-methyl-D-glucose than of L-glucose.