Acrosome status in human spermatozoa from 20 normozoospermic men was evaluated by flow cytometry following the induction of the acrosome reaction with the ionophore A23187. Dual fluorescence staining of methanol fixed spermatozoa incubated with and without (control) the ionophore A23187 was performed with probes which targeted the outer acrosomal membrane (OAM) (rhodamine-labelled Arachis hypogaea agglutinin) or constituents of the acrosomal vesicle (fluorescein-labelled Pisum sativum agglutinin). Flow cytometry analysis revealed two major subpopulations of cells: acrosome-intact and acrosome-reacted spermatozoa after induction of the acrosome reaction. The intensity of green and red fluorescence in acrosome-reacted spermatozoa was significantly lower than that of the acrosome-intact control spermatozoa (P < 0.0001). The intensity of green fluorescence in the acrosome-intact subpopulation of spermatozoa was significantly higher than that of the control population (P < 0.002). Exposure of spermatozoa to the ionophore A23187 resulted in reliable enhancement of the number of spermatozoa with very high intensity of green and/or red fluorescence compared with the control (P < 0.03). An inverse correlation between the number of acrosome-reacted spermatozoa and spermatozoa with a very high intensity of green and/or red fluorescence was demonstrated (r = -0.631, P < 0.01). This method provides an objective and efficient procedure for quantitative estimation of the acrosomal status of human spermatozoa.