AbstractBackground: Characteristic membrane changes in spermatozoa culminating in acrosome reaction and sperm‐egg fusion, and suspected involvement of actin‐containing cytoskeleton in membrane changes in general, prompted us to investigate subcellular distribution of actin and actin‐binding proteins in bovine spermatozoa subjected to various extractions which sequentially denude the sperm investments.Methods: Spermatozoa were treated with either 1% SDS, 0.1% Triton X‐100, 0.1% Hyamine, or 1 M MgCl2or were sonicated. Immunostaining of actin, α‐actinin, spectrin, and acrosin as well as electron microscopic analysis of extracted spermatozoa were carried out.Results: Extractions caused evagination of the acrosomal lamina which retained focal contacts with the inner acrosomal membrane. Extractions further revealed lateral prongs at the anterior border of the postacrosomal sheath. Labeling for α‐actinin and spectrin was localized in the acrosinpositive acrosomal lamina, neck, and principal piece, the latter containing also relatively extraction‐resistant oligomeric or polymerized actin. In the postacrosomal area, actin was accumulated in the extraction‐resistant posterior ring structure and anteriorly at the sites apparently related to the lateral prongs. Notably, spectrin reactivity was enhanced by MgCl2in head, neck, and principal piece, and sonication abolished cytoskeletal immunoreactivity in the head.Conclusions: Destabilization of membranes with selected extractions induces changes in the acrosomal lamina mimicking acrosomal vesicle formation. The lateral prongs and posterior ring structure, respectively, may serve as anterior and posterior anchors for the extraction‐resistant post‐acrosomal sheath. The lateral prongs may also be a merger zone for actin, α‐actinin, and spectrin with important implication on sperm function. The latter two proteins may be involved in acrosomal vesicle formation. It is apparent that extractions have a significant effect on the detectability of sperm cytoskeletal elements