The facilitated diffusion glucose transporter in the plasma membrane of intact outer segments isolated from bovine retinal rods (ROS) was characterized by measurements of: (1) 14C-labeled 3-O-methylglucose fluxes; and (2) glucose-sensitive binding of 3H-labeled cytochalasin B to ROS membranes. Inhibition of 3-O-methylglucose influx into ROS, inhibition of 3-O-methylglucose efflux from ROS and glucose-sensitive binding of cytochalasin B to ROS showed very similar cytochalasin B inhibition/dissociation constants of 0.9 microM, 1.3 microM and 1.3 microM, respectively. D-glucose inhibited both 14C-labeled 3-O-methylglucose transport and cytochalasin B binding. The above results suggest that D-glucose-sensitive cytochalasin B binding reflects specific binding to the ROS glucose transporter and the density of glucose transporter in the ROS plasma membrane was determined to be 800 microns-2, comparable to relatively abundant ROS plasma membrane proteins such as the cGMP-gated channel and the Na-Ca+K exchanger. Displacement of 3H-labeled cytochalasin B by non-transportable hexoses was used to localize the hexose transporters to the ROS plasma membrane and to examine a simple single-site, alternating conformation model for hexose transport. A comparison between the Glut1 glucose transporters of bovine ROS, bovine erythrocytes and human erythrocytes suggests that kinetic and pharmacological characteristics of glucose transporters cannot be predicted in a simple manner from gene type and species.