A major mechanism regulating CYTase, the rate-limiting enzyme in phosphatidylcholine (PC) synthesis, involves lipid-dependent translocation of inactive soluble CYTase (L-form) to membranes where the enzyme is activated. In yeast, the phosphatidylinositol transfer protein SEC14p regulates golgi CYTase activity, but the mechanisms that regulate the translocation/activation of CYTase in mammalian cells have not been completely elucidated. The purpose of this study was to determine whether mammalian PI-TP regulates CYTase in the lung. In cytosol from the MLE12 rat respiratory epithelial cell line, PI-TP co-localized with CYTase L-form fractions. We studied the effect of immunodepleting PI-TP (using anti-PI-TP antisera from GM Helmkanp, U. Kansas) on L-form CYTase activity. PI-TP depletion did not co-precipitate CYTase, but reduced lipid-independent CYTase activity in L-form fractions 3-4.5-fold. To determine the effect of immunodepleting PI-TP on oleic acid (OA)-induced translocation and activation of CYTase in post-mitochondrial supernatants(PMS), PI-TP-depleted and sham-depleted PMS were incubated with 250 μM oleic acid (OA) at 37°C for 20 min and were fractionated on 7.2-36% glycerol gradients. CYTase was analyzed in each fraction by functional assay and immunoblotting. In the absence of OA treatment, 17% of the total CYTase activity was present in the microsomal pellet. OA treatment increased the microsomal CYTase activity to 72.9±18.4% of total CYTase activity in the sham-depleted PMS, but the microsomal CYTase activity increased to only 41.9±17.6% of total activity in the PI-TP-depleted PMS. By contrast, immunoblotting demonstrated 2 to 23-fold more CYTase protein in PI-TP-depleted vs. sham-depleted PMS. Thus, while OA-stimulated translocation of CYTase protein from cytosol to microsomes was higher in the PI-TP-depleted PMS, the specific activity of the translocated enzyme was reduced over 15-fold compared with PI-TP-containing PMS. These data suggest an essential function for PI-TP in the coordinated translocation and activation of CYTase.