The acrosome reaction in guinea-pig sperm is accompanied by a marked cavitation of the acrosomal contents. Two divergent views are held as to whether this cavitation precedes or follows the membrane fusion that occurs in the reaction. To distinguish between these 2 views cavitation was induced in media containing a colloid, either Ficoll 70 or inulin, either by inducing a normal acrosome reaction using the calcium ionophore A23187 or by using the detergent Triton X100. Both Ficoll 70 and inulin, when incorporated into media of normal osmolality, were able to suppress various features of the cavitation. Complete retention of acrosomal shape was achieved in sperm treated with detergent in 30% (W/V) Ficoll 70 solution despite the absence of the limiting acrosomal and plasma membranes. This evidence supports the suggestion that the cause of the cavitation is a colloid osmotic pressure within the acrosomal matrix. This in turn supports one of the 2 proposed mechanisms for the temporal sequence of events occurring in the acrosome reaction.