T o determine whether basal phosphoinositide turnover plays a role in metabolic regulation in resting rabbit aortic intima-media incubated under steady state conditions, we used deprivation of extracellular myo-inositol as a potential means of inhibiting basal phosphatidylinositol (PI) synthesis at restricted sites and of depleting small phosphoinositide pools with a rapid basal turnover. Medium myo-inositol in a normal plasma level was required to prevent inhibition of a specific component of basal de novo PI synthesis that is necessary to demonstrate a discrete rapidly turning-over [1,3-14C]glycerol-labeled PI pool. Medium myo-inositol was also required to label the discrete PI pool with [1-14C]arachidonic acid (AA). The rapid basal turnover of this PI pool, when labeled with glycerol or AA, was not attributable to its utilization for polyphosphoinositide formation, and it seems to reflect basal PI hydrolysis. Depleting endogenous free AA with medium defatted albumin selectively inhibits the component of basal de novo PI synthesis that replenishes the rapidly turning-over PI pool. A component of normal resting energy utilization in aortic intima-media also specifically requires medium myo-inositol in a normal plasma level and a free AA pool; its magnitude is unaltered by indomethacin, nordihydroguaiaretic acid, or Ca2+-free medium. This energy utilization results primarily from Na+/K+ ATPase activity (ouabain-inhibitable O2 consumption), and in Ca2+-free medium deprivation of medium myo-inositol or of free AA inhibits resting Na+/K+ ATPase activity to a similar degree (60%, 52%). In aortic intima-media basal PI turnover controls a major fraction of resting Na+/K+ ATPase activity.