Once infected by HIV-1, microglia abundantly produce accessory protein Nef that enhances virus production and infectivity, but little is known about its intracellular compartmentalization, trafficking mode(s), and release from microglia. Here, we transfected immortalized human microglia with a plasmid encoding Nef tagged with green fluorescent protein (Nef.GFP) to optophysiologically identify Nef.GFP-associated cellular compartments and examine their mobility and Nef release from cultured cells. As revealed by analysis of confocal micrographs, Nef.GFP-positive structures were smaller than dextran-laden vesicles, and displayed slow and non-directional mobility, in contrast to the faster and directional mobility of dextran-laden vesicles. Nef.GFP negligibly co-localized with different endosomes including Dextran-laden vesicles, but significantly co-localized with membranes immuno-positive for tetraspanins CD9 (36±4 %) and CD81 (22±1 %). Expression of Nef was further accompanied by a decrease in number of CD9- and CD81-positive structures in the periplasmalemmal space of microglia. Ionomycin-evoked elevation in cytosolic calcium activity negligibly affected mobility of Nef.GFP structures, but strongly and irrecoverably attenuated mobility of dextran-laden vesicles. A slow time-dependent decrease in number of Nef.GFP-positive structures was observed in non-stimulated controls (5±1 structures/min), but not in ionomycin-stimulated cells (0±2 structures/min; P<0.05), indicating that elevated free intracellular Ca2+ concentration ([Ca2+]i) inhibits the release of Nef.GFP structures. Our results thus suggest that Nef is likely released by ectosomes after budding from the plasmalemma and not by regulated exocytosis of exosomes from the lumen of multivesicular bodies in microglia.