In isolated rat adipocytes, basal as well as insulin-stimulated 3-O-methylglucose transport was inhibited nearly completely (maximal inhibition: 95%) by the nucleoside transport inhibitors dipyridamole (IC50 = 5 microM), nitrobenzylthioguanosine (20 microM), nitrobenzylthioinosine (35 microM) and papaverine (130 microM). Transport kinetics in the presence of 10 microM dipyridamole revealed a significant increase in the transport Km value of 3-O-methylglucose (3.45 +/- 0.6 vs 2.36 +/- 0.29 mM in the controls) as well as a decrease in the Vmax value (4.84 +/- 0.95 vs 9.03 +/- 1.19 pmol/s per microliter lipid in the controls). Half-maximally inhibiting concentrations of dipyridamole were one order of magnitude higher than those inhibiting nucleoside (thymidine) uptake (0.48 microM). The inhibitory effect of dipyridamole (5 microM) reached its maximum within 30 s. The agent failed to affect insulin's half-maximally stimulating concentration (0.075 nM) indicating that it did not interfere with the mechanism by which insulin stimulates glucose transport. Further, dipyridamole fully suppressed the glucose-inhibitable cytochalasin B binding (IC50 = 1.65 +/- 0.05 microM). The data indicate that nucleoside transport inhibitors reduce glucose transport by a direct interaction with the transporter or a closely related protein. It is suggested that glucose and nucleoside transporters share structural, and possibly functional, features.