Filipin, a polyene antibiotic, fluoresces and forms 15-25 nm aggregates when combined with beta-hydroxysterols, rendering sterols detectable by fluorescence microscopy and by electron microscopy of thin sections and freeze-fracture replicas. We applied filipin in a glutaraldehyde fixative to tissue-cultured cells of Drosophila melanogaster larvae, in which sterol concentration can be regulated. Since the number of filipin-sterol aggregates observed in membranes was found to be proportional to the amount of sterol experimentally inserted, utilizing filipin is a valid method for quantifying, as well as for mapping, sterol distribution in biological membranes. Other antibiotics may be similarly used for localizing some species of negatively charged phospholipids. In addition to cytochemical identification of specific lipids, rapid freezing and deep etching of unfixed, non-cryoprotected cells may permit us to examine membrane lipids in different physical states: liquid-crystalline and gel. Combining these several techniques has resulted in new data concerning the disposition of lipids during the intimate juxtaposition of membranes preceding fusion. For example, in guinea-pig sperm, foci of closely apposed membranes are bereft of beta-hydroxysterols and intramembranous particles. Such regions of membrane sometimes exist in a crystalline state and may be rimmed by negatively charged phospholipids. As previously noted in other areas of cytochemistry, the in situ localization of specific substances provides information unobtainable by morphological or biochemical techniques alone.