Electron microscopy (EM) of freeze-fractured membranes provides more information about internal membrane structure than EM of thin-sectioned or negatively stained material. However, it has heretofore been impractical to use freeze fracture routinely for analysis of highly purified membrane fractions obtainable in small (micrograms) amounts, because the technique, when conventionally applied to minute pellets, yields only one fracture of unpredictable quality; it also precludes in parallel biochemical studies by using up the entire preparation. To solve this problem, we have developed a method for freeze fracturing tiny droplets of suspended membranes containing 1-10 micrograms membrane protein, thereby allowing both multiple fractures and biochemical studies. Before fracture, the final membrane fractions can be concentrated, subjected to experimental manipulations, cross-linked, and glycerinated in a dialysis bag. The technique is illustrated on isolated gap junctions from rabbit hearts, which were chosen because their unique internal membrane structure allows unequivocal identification of membrane type based on structural criteria.