The effects of three membrane-active agents, filipin, digitonin, and polymyxin B on the plasma membrane of ram spermatozoa have been studied by freeze-etch electron microscopy. Filipin and digitonin both reacted with cholesterol and caused visible membrane modification in cholesterol-rich regions, with filipin being a more specific agent than digitonin. Polymyxin B, which is known to interact specifically with negatively charged phospholipids of bacterial membranes, exhibited a selective binding and subsequent modification of sperm plasma membranes. This binding was shown to be inhibited in the presence of 1 mmol/1 Ca2+. We hence propose that both filipin and polymyxin B are useful cytological markers for specific biological molecules in eukaryote membranes--filipin for cholesterol, and polymyxin B for negatively charged phospholipids.