Experiments were conducted to test the hypothesis that a longitudinal gradient in myogenic responsiveness exists within an arteriolar network. Single arterioles were dissected from the hamster cheek pouch, cannulated with micropipettes, and transferred to an inverted microscope for in vitro study. Pressure-diameter relationships of five branching orders of arterial vessels were measured in the presence of spontaneous vascular tone and after elimination of tone with a Ca(2+)-free solution containing nitroprusside. At luminal pressures matching those found in vivo, the diameters of the vessels with spontaneous tone were as follows: small arteries, 81 microns; first-order arterioles, 52 microns; second-order arterioles, 32 microns; third-order arterioles, 24 microns; and fourth-order arterioles, 11 microns. All branching orders of vessels exhibited true myogenic responses as indicated by negative slopes of their pressure-diameter relationships. Each vascular branching order exhibited its maximum myogenic responsiveness at a pressure near or just slightly higher than its normal pressure as measured in vivo. Relative myogenic responsiveness increased with decreasing vessel size down to the level of the second- and third-order arterioles, whereas fourth-order arterioles were substantially less responsive than third-order arterioles. A compilation of data from numerous in vivo and in vitro studies suggests that the same myogenic response pattern may be found in other vascular beds.