A comparative analysis has been undertaken of the behaviour of probes targeting the outer acrosomal membrane (Arachis hypogaea lectin) or constituents of the acrosomal vesicle (Pisum sativum lectin or monoclonal antibody CRB9) following the induction of the acrosome reaction in human spermatozoa with the ionophore A23187. The results obtained with these two classes of reagent were highly correlated (r = approximately 0.8), although the absolute rates of acrosome reaction were significantly different; the probe targeting the outer acrosomal membrane (A. hypogaea) consistently gave higher results than either of the probes targeting the acrosomal contents. Time-dependent studies, employing a double-labelling technique, indicated that this difference was apparent from the earliest time point examined (15 min after A23187 addition) and reflected the more rapid dissipation of the A. hypogaea lectin from the acrosomal region of the cell than either of the probes targeting the acrosomal vesicle. These results indicate that the outer acrosomal membrane is dispersed from acrosome-reacting human spermatozoa more rapidly than certain major constituents of the acrosomal vesicle and have possible implications for the design of diagnostic assays focusing on this aspect of human sperm function.