In the tammar wallaby (Macropus eugenii), post-testicular acrosomal shaping involves a complex infolding and fusion of the anterior and lateral projections of the scoop-shaped acrosome into a compact button-like structure occupying the depression on the anterior end of the sperm nucleus. The present study has generated cytochemical and histological evidence to demonstrate that the occurrence of actin filaments (F-actin, labelled by Phalloidin-FITC) in the acrosome of tammar wallaby spermatozoa is temporally and spatially associated with the process of acrosomal shaping in the epididymis, through a pool of monomeric actin (G-actin, labelled by Rh-DNase I) present in the acrosome throughout all stages of epididymal maturation. F-actin was not detected in the acrosome of testicular spermatozoa, but was found in the infolding and condensing acrosome of caput and corpus epididymal spermatozoa. When the spermatozoa completed acrosome shaping in the cauda epididymidis, F-actin disappeared from the acrosomal area. The strong correlation between the occurrence of F-actin and the events of acrosomal shaping suggested that the post-testicular shaping of the acrosome might depend on a precise succession of assembly and disassembly of F-actin within the acrosome as the spermatozoa transit the epididymis. Thus, actin filaments might play a significant role in the acrosomal transformation, as they are commonly involved in morphological changes in somatic cells.