The aim of this study was to investigate basically, using the gametes of golden hamster, whether acridine orange (AO) fluorescent dye was efficient for detecting the sperm-egg fusion in vitro, and to evaluate the usefulness of this dye by comparison with other staining methods such as toluidine blue and Giemsa. Before insemination with zona-free hamster mature oocytes (metapase-II), the nuclei of acrosome reacted spermatozoa collected from the cauda epididymis emitted bright green AO fluorescence. Chronologically sperm nuclei changed AO fluorescence from green to red before it began to decondense within the ooplasm. Condensed nuclei attached to the oolemma of GV oocytes and pronuclear stage eggs, which were thought to be fused, were stained red 1 hour after insemination. On the other hand, although the spermatozoa incubated with zonafree, metaphase-II oocytes under calcium-magnesium free conditions had condense shaped nuclei after 1 hour insemination, the AO stained nuclei were completely green. The AO staining indicates the thiol-disulfide status of sperm nuclei, namely, green and red fluorescences mean S-S rich and S-S poor, respectively. Therefore, since nucleoproteins of the sperm nucleus should be reduced after being mixed with the ooplasma during the sperm-egg fusion event, the condense shaped and red nuclei are considered fused with oolemma before nuclear decondensation occurred. The AO fluorescence dye was proven to be effective compared with other staining methods reported previously, and AO staining will be useful for a precise and efficient means of detecting and investigating sperm-egg fusion events in mammals.