Smooth muscle responses to Na+ pump inhibition are thought to reflect two elements: a neurogenic contribution, involving catecholamine release from nerve terminals, and a myogenic response, attributed to relations between pump activity, [Na+]i, and [Ca2+]i. In the present study, we describe the time course and magnitude of cell Na+ changes, assessed by two methods, atomic absorption and nuclear magnetic resonance spectroscopy during the myogenic contractile response of rabbit aorta strips to ouabain. A threshold concentration of 3±10−7 mol/L induced a gradual rise in [Na+]i. Both methods showed an essentially identical monotonic rise over 4 to 8 hours from a baseline level of 8 to 10 mmol/L water to a peak, which was approximately fivefold higher. The neurogenic (rapid) and myogenic (delayed and gradual) contractile responses were temporally distinct. Ouabain at 10−7 mol/L, a concentration 10- to 100-fold lower than the threshold for catecholamine-dependent rapid-onset responses, induced only a delayed and gradual contractile response, which reached a maximum at 6 to 8 hours. With 10−6 mol/L ouabain, the delayed response of 1.6±0.2 g peaked at 7.3±1.1 hours and was sustained for 16 hours. The time course was similar to that for change in [Na+] but somewhat later. Ouabain at 10−5 and 10−4 mol/L induced a delayed response that was identical in magnitude but also induced an early rapid contractile response, which was prevented by reserpine or phentolamine pretreatment. These agents did not influence the delayed response. Reduction of the bath [Na+] to 25 mmol/L by replacing sodium chloride with choline chloride abolished the delayed contractile response to 10−6 mol/L ouabain. In parallel experiments, a fall in 86Rb uptake occurred within 15 minutes after exposure to ouabain; thus, limited diffusion of ouabain was not responsible for the delayed contractile response. The Na+ content and myogenic response of vascular smooth muscle to Na+ pump inhibition have a similar time course, gradual in onset and sustained for hours, in accord with models implicating intracellular Na+ in the vascular smooth muscle myogenic contractile response to Na+ pump inhibition.