[3H]Cytochalasin B binding and its competitive inhibition by D-glucose have been used to quantitate the number of functional glucose transport units in plasma and microsomal membranes prepared from intact rat diaphragm. In a series of three experiments, plasma membranes prepared from diaphragms which have not been incubated with insulin bind approximately 16 pmol of cytochalasin B/mg of membrane protein to the D-glucose-inhibitable binding site. If 280 nM (40,000 microunits/ml) insulin is present during the incubation, cytochalasin B binding to the plasma membranes is increased approximately 2-fold without alteration in the dissociation constant of this site. Membranes in the microsomal fraction prepared from diaphragms which have been incubated for 30 min in the absence of insulin contain 21 pmol of D-glucose-inhibitable cytochalasin B binding sites/mg of membrane protein. However, in the presence of insulin during the incubation period, the number of these sites in the microsomal fraction is decreased to 12 pmol/mg of membrane protein. These results suggest that insulin stimulates glucose transport in the isolated rat diaphragm primarily through a translocation of functional glucose transport units from an intracellular membrane pool to the plasma membrane. These results are similar to the results observed in rat adipose cells (Cushman, S. W., and Wardzala, L. J. (1980) J. Biol. Chem. 255, 4758-4762) and suggest that this mechanism of insulin-stimulated glucose transport activity may be general to other cell types.