The spermatozoa of most marsupials lack nuclear stabilising disulfide-bonded protamines found in eutherian mammals. However, disulfide stabilisation has been observed in the acrosome of macropodid (Macropus eugenii) and phalangerid (Trichosurus vulpecula) marsupials. As a result this organelle, which is normally fragile in eutherian mammals, is robust and able to withstand physical and chemical challenge in these marsupials. The present study examined acrosomal characteristics of the spermatozoa of three dasyurid marsupials; the fat-tailed dunnart (Sminthopsis crassicaudata), eastern quoll (Dasyurus viverrinus) and northern quoll (Dasyurus hallucatus). In all species examined Bryan’s staining demonstrated that significant acrosomal loss occurred following physical challenge with osmotic stress, cryopreservation without cryoprotectant and exposure to detergent (Triton-X). Bromobimane staining indicated that the acrosomes of dasyurids lacked stabilising disulfide bonds. As reported for the wallaby and possum, calcium ionophore (A23187) did not induce the acrosome reaction-like exocytosis in dasyurid spermatozoa but treatment with diacylglycerol (DiC₈) caused significant acrosome loss at concentrations similar to those effective for other marsupials. The present study found that the spermatozoa of dasyurids are more sensitive to physical challenge than the previously-studied marsupials and we suggest that this is due to the absence of acrosomal stabilising disulfide bonds.