Triacylglycerol feeding increases serum intestinal alkaline phosphatase (IAP) activity and leads to the appearance of an alkaline phosphatase-containing particle in the luminal washings over the apical surface of the rat enterocyte and in the blood (J. Clin. Invest. 84: 1355-1361, 1989). To examine the coordinate appearance of these particles and the enzyme and to follow their distribution in vivo after feeding, an enzyme-linked immunoabsorbent assay (ELISA) was developed, using antisera raised against the purified intact surfactant-like particle. Tissue compartments that were examined for phosphatase activity and particle content included isolated enterocytes, lamina propria, intestinal luminal washings, and serum. Alkaline phosphatase activity peaked earliest in the lamina propria (3 h), followed by the enterocyte and the luminal washings (5 h) and serum (7 h). Surfactant-like particle content peaked in the enterocyte and lamina propria at 3 h, followed by the serum (3-5 h) and the luminal washings (5 h). The buoyant density of the particle in the enterocyte (d = 1.08-1.09) and serum (d = 1.07-1.08) after fat feeding was similar to that of the isolated particle (d = 1.07-1.08). The density of particle proteins detected by ELISA in fasted serum was more diffuse and >1.10, consistent with partial degradation of the particle and/or its proteins. These data confirm that the particle and its bound IAP are secreted from the enterocyte after triacylglycerol feeding and that they appear in compartments adjacent to both the basolateral (serum) and apical (luminal wash) surfaces of the enterocyte. Moreover, the data show that the site of initial peak appearance of extracellular particle is the lamina propria, followed by peak concentration of the particle in both the serum and on the luminal surface. The lamina propria may play a physiological role, perhaps as reservoir and/or as filter in the extracellular processing of the surfactant-like particles.