Recent studies indicate that subacrosomal proteins are necessary for the attachment of the acrosome onto the nucleus during sperm formation, and for the stability of the nuclear membrane during fertilization. For the first time, subacrosomal proteins have been isolated from a marsupial species, the tammar wallaby (Macropus eugenii), using a method developed in our laboratory. Whole ejaculated spermatozoa were fractionated into head and tail sections by ultrasonication to extract subacrosomal proteins. The heads (> 95% purity) were then isolated from tail sections using centrifugation with a three-step discontinuous sucrose gradient (35, 68 and 75% (w/v). The heads were treated with 0.1% (v/v) Triton X-100 which stripped off the acrosome, but not the subacrosomal proteins, from the head. The proteins were finally extracted by 100 mmol NaOH l-1. Four prominent subacrosomal polypeptides, with molecular masses of 45, 38, 33 and 29 kDa, were recognized from the SDS-PAGE gel. The localization of these polypeptides (particularly the 45 kDa polypeptide) was confirmed by fluorescent and immunogold labelling with polyclonal antibodies raised in mice against the obtained polypeptides. In wallaby testes, the 45 kDa polypeptide was detected as early as at the step 3 spermatid and was mainly associated with the membrane of the newly formed acrosome vesicle. This polypeptide was also found on the acrosomal membrane of all older spermatids. The 45 kDa polypeptide was found on the acrosomal region of the spermatozoa collected from the caput, corpus and cauda of the epididymis. The similarity of the sperm anatomy of the tammar wallaby with that of other marsupials, such as the brushtail possum, implies that this procedure could be applied effectively to other marsupial species with minor modification.