The disposition of spermatozoa in the female tract of the dasyurid marsupial, Sminthopsis crassicaudata, was examined before and after ovulation, by transillumination using differential interference optics. At 6-30 h after mating in females with maturing follicles, spermatozoa were restricted to special storage crypts that define the isthmus of the Fallopian tube. These spermatozoa were mostly immotile and were spear shaped, except for 50-200 vanguard spermatozoa within anterior crypts that were T shaped due to a rotation of the head on the tail. After ovulation and arrival of eggs in the narrow upper ovarian segment of the tube, several hundred isthmic spermatozoa migrated to that region, dispersed singly or in small groups of variable motility with some festooned around eggs. Most clearly displayed a T configuration, and subsequently, those remaining in the isthmic crypts also adopted a T shape in situ. The motility of mature epididymal spermatozoa and of those flushed from the isthmus before and after ovulation was similar in vitro, and all were spear shaped except for some T-shaped isthmic spermatozoa recovered after ovulation. However, neither spear- nor T-shaped active isthmic spermatozoa ever adhered to the zona pellucida when incubated with eggs. We conclude that transport to and storage of spermatozoa in the isthmus of the Fallopian tube, and their later migration up to the fertilization site and coincident transformation to a T shape, all are closely regulated by the ovarian follicular cycle in Sminthopsis. Only on reaching the upper ovarian segment of the Fallopian tube do Sminthopsis spermatozoa appear to be able to bind to the egg, when the T shape brings the whole surface area of the acrosome into apposition with the zona pellucida. These observations are discussed with special reference to sperm capacitation, sperm penetration mechanisms, and the different idiosyncratic designs of the sperm head in marsupial and eutherian mammals, respectively.