The present study aimed to investigate the effects of cytochalasin B (20 μM) on the uptake of 3-O-[14C]-methyl-D-glucose or D-[U-14C]glucose (8.3 mM each) by BRIN-BD11 cells. Taking into account the distribution space of tritiated water (3HOH), which was unexpectedly increased shortly after exposure of the cells to cytochalasin B and then progressively returned to its control values, and that of L-[1-14C]glucose, used as an extracellular marker, it was demonstrated that cytochalasin B caused a modest, but significant inhibition of the uptake of D-glucose and its non-metabolized analog by the BRIN-BD11 cells. These findings resemble those observed in acinar or ductal cells of the rat submaxillary gland and displayed a relative magnitude comparable to that found for the inhibition of D-glucose metabolism by cytochalasin B in purified pancreatic islet B cells. These findings reinforce the view that the primary site of action of cytochalasin B is located at the level of the plasma membrane.