Immunocytochemistry was used to determine the intracellular location of perilipins in adipocytes and the occurrence of these proteins in tissues involved in triacylglycerol metabolism. Confocal microscopy and 3-dimensional analysis of 3T3-L1 adipocytes showed that perilipin immunofluorescence, present on the surfaces of all sized lipid droplets, appeared unevenly dispersed on the surfaces of many large lipid droplets. Electron microscopy revealed that immunogold staining for perilipin was located directly on the surface layer apposed to and surrounding the core triacylglycerol of intracellular lipid droplets of adipocytes in culture or from white and brown adipose tissue. Freeze-fracture electron microscopy indicated that the hydrophobic face of this surface monolayer contained particles identical in size and distribution to intramembranous particles (IMPs), which are unique structural features of the hydrophobic faces of bilayered membranes. Also, freeze-fracture replicas revealed areas of continuity between the surface layer of lipid droplets and the membrane leaflets of endoplasmic reticulum, suggesting that the droplet monolayer surface is an area of endoplasmic reticulum membrane leaflet modified by its unique content of perilipin. Microperoxisomes, identified by immunostaining for catalase, were found closely associated with lipid droplets, but external to and not in contact with the lipid droplet surface layer. Vimentin, identified by immunofluorescence, was present around the periphery of most lipid droplets in 3T3-L1 cells during early stages of adipocyte development but, in contrast to perilipins, vimentin was not around the periphery of many large lipid droplets in mature cells. Although perilipin was at the surface of lipid droplets in adipocytes of lactating mammary gland, none was found to be associated with the milk lipid droplets in alveolar epithelial cells, nor was the protein found on the surfaces of lipid droplets in hepatocytes. Studies in mammary gland show that perilipin immunostaining will be a valuable tool for the identification of tissue adipocytes severely depleted of their triacylglycerol stores and thus without their characteristic spherical shape. Perilipin's singular location on the surface monolayer of intracellular lipid droplets supports an intimate role for the protein in the triacylglycerol metabolic functions of adipocytes.