Phosphatidylinositol (PI), mainly stearoyl-arachidonyl PI, occurs as a minor phospholipid constituent in both chyle and plasma lipoproteins. The kinetics and the pathway by which plasma and chyle PI is metabolized have not been investigated. The role of lipoprotein PI in the supply of arachidonic acid (20:4) and inositol lipid components to different tissues is thus unknown. In this study we examined the fate of chyle PI in vivo and its hydrolysis by lipoprotein lipase (LPL), hepatic lipase (HL), and postheparin plasma in vitro. Chyle and chylomicrons were labeled in the PI portion by feeding [3H]myo-inositol and in the phosphatidylcholine (PC) portion by feeding [14C]choline in a linoleate-rich fat meal (Intralipid) to mesenteric duct-cannulated rats. After intravenous injection of doubly labeled chyle into normal rats, [3H]PI disappeared from plasma at a slower rate than [14C]PC; after 60 min 41.6 +/- 2.7% 3H and 24.3 +/- 1.8% 14C (means +/- SEM, n = 4, P < 0.01) remained in plasma lipids. About 15% of both isotopes were in liver lipids after 60 min. Previous injection of a blocking antiserum against rat HL did not significantly influence the serum and liver radioactivity after 60 min. Radioactive PI was rapidly transferred to high density lipoproteins (HDL) during the metabolism of chylomicrons. Analysis of 3H and 14C in different molecular species of PI and PC in chyle and in serum indicated that there was no significant difference in disappearance rates between various species, stearoyl-arachidonyl PI thus disappearing at the same rate as total [3H]PI. Both lipoprotein lipase (LPL) and HL catalyzed formation of lyso-PI in vitro, the rate being increased by the addition of serum. About 60% of the lyso-PI formation catalyzed by postheparin plasma in 60 min could be blocked by antiserum to HL, which almost completely blocked the hydrolysis occurring after the first 10 min. The study thus shows that both LPL and HL hydrolyze chylomicron PI in vitro. LPL and HL may, however, be of limited importance for the clearance of chyle PI in vivo, most of the chylomicron PI being transferred to HDL, and thereafter eliminated from plasma at a slow rate mainly by other mechanisms.