A procedure was developed to evaluate mouse sperm acrosomal status and viability simultaneously utilizing flow cytometry. Four fluorescein isothiocyanate (FITC)-conjugated lectins, peanut agglutinin (PNA), concanavalin agglutinin (ConA), Pisum sativum agglutinin (PSA), and soybean agglutinin (SBA), were investigated, with PNA providing the greatest sensitivity and specificity in distinguishing acrosome-present and acrosome-absent mouse spermatozoa. To expose lectin binding sites, digitonin (20 μM at room temperature for 10 min) was used to permeabilize sperm plasma membranes. Sperm cell viability was determined by Hoechst 33258 (H258) exclusion. To prevent permeabilized cells from staining with H258, salmon sperm DNA (SS-DNA) was applied to bind excess dye in the solutions after supravital staining. Calcium ionophore (A23187; 5 or 20 μM) was used to induce acrosome reactions. The results of flow cytometric analyses were compared with epifluorescence microscopic observation and were highly correlated (r = 0.999; P < 0.001). The method developed provides an objective and efficient procedure to estimate simultaneously both acrosomal status and viability of mouse spermatozoa. © 1993 Wiley-Liss, Inc.