Beta-Neurotoxins from certain snake venoms are highly specific toxins acting at the presynaptic side of the neuromuscular junction. In this study biochemical aspects of this high specificity have been investigated. When toxins (notexin and Naja nigricollis basic phospholipase) act on a mixture of subcellular fractions obtained from brain cortex (synaptosomes, myelin, and mitochondria), the synaptosomal fraction is preferentially attacked and shows the highest release of membrane protein. As seen from isolated fractions, however, even the mitochondria are rapidly and strongly attached. Examining the phospholipase A2 activity of the toxin instead of the release of proteins reveals that synaptosomes represent the best substrate. In contrast to nonneurotoxic phospholipases A2, that from neurotoxin preferentially uses synaptosomal phosphatidylcholine as a substrate when pure phospholipids isolated from subcellular fractions are used. A relationship between the cholesterol/phospholipid ratio and the sensitivity to toxin action in the various subcellular fractions was found. These data suggest that the neurotoxic effect is mainly due to the substrate specificity of the beta-neurotoxins. It is suggested that synaptosomal phosphatidylcholine, embedded in a membrane containing a low amount of cholesterol, is a highly specific substrate for beta-neurotoxins.