The results of the present work provide support for the utility of studying [<sup>3</sup>H]- ouabain binding to in vitro segments of vascular smooth muscle as an indication of the number of Na-K pump sites in blood vessels of rats. Specific binding of [<sup>3</sup>H]-ouabain to strips of rat abdominal aorta and segments of rat caudal artery was saturable and reversible. Specific [<sup>3</sup>H]-ouabain binding was displaced by several cardiac glycosides but not by steroid hormones or sodium vanadate. The dissociation constant (K<sub>D</sub>) of [<sup>3</sup>H]-ouabain binding, as determined by equilibrium and kinetic studies, was in the range of 50–150 n M in these vessels. Scatchard analysis indicated that the maximum number of [<sup>3</sup>H]-ouabain-binding sites (B<sub>max</sub>) was 20 and 46 fmol/mg wet weight in the aorta strips and caudal artery, respectively. A comparison of [<sup>3</sup>H]-ouabain binding in caudal arteries of spontaneously hypertensive rats and of age-matched, genetic controls indicated that neither the affinity for binding nor the maximum number of binding sites was different. This suggests that this genetic form of hypertension is not associated with an alteration in the number of vascular Na-K pump sites.