When guinea pig spermatozoa were preincubated 1 hr in Ca²⁺‐free medium containing dilysocardiolipin (100–125 μg/ml) then exposed to Ca²⁺, the majority underwent an immediate acrosome reaction. Monolyso‐cardiolipin was much less effective and the native cardiolipin was totally ineffective. Some fatty acids added to the medium could also render the spermatozoa capable of undergoing their acrosome reactions, arachidonic acid in methyl ester form being most potent. It is known that sperm membrane contains phospholipase A which cleaves membrane phospholipids into lysophospholipids and fatty acids. Most lysophospholipids are known to be potent acrosome reaction–promoting agents. As some forms of fatty acids are also potent acrosome reaction–promoting agents, both products of membrane phospholipid hydrolysis by phospholipase A (i.e., both fatty acids and lysophospholipids) may work synergistically to effect the conversion of stable sperm membranes to a fusion competent state capable of engaging in the acrosome reaction.