Renal phospholipid metabolism was studied in rat cortical tubule suspensions by combining net measurements with precursor incorporation studies in order to be able to calculate the turnover of the different phospholipid species. Net amounts of tubular phosphatidylcholine (PC), phosphatidylinositol (PI) and phosphatidylethanolamine (PE) were 96.7 +/- 3.9, 29.2 +/- 2.9 and 74.1 +/- 6.0, mumol per g protein, respectively. Incubation of tubules in the absence or presence of renal substrates did not change net phospholipid contents. The predominant fatty acids were palmitate (47%) in PC and stearate in PI (56%) and PE (41%). Highest [14C]palmitate and [14C]glycerol incorporation rates were found in PC. In contrast, 32P-labeling was highest in phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate exceeding PI-labeling by a factor of 2.4 and 5.2, respectively. In contrast, [3H]inositol incorporation into phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate was only 20% compared to that into PI. Addition of renal substrates like lactate, glutamine, glycerol and fatty acids increased precursor incorporation. The stimulating effects of gluconeogenic precursors and fatty acids were more than additive. Comparison of rates of [14C]glycerol, [32P]- and [3H]inositol incorporation suggests that de novo phospholipid biosynthesis is represented by these measurements, while the fatty acid fraction in addition turns over by exchange processes. According to our conclusions PE, PC, PI and phosphoinositides turn over with a half-life of 139, 16, 15 and 0.7 h, respectively, under optimal in vitro conditions.