Maturation of marsupial oocytes in vitro, an important step in the analysis of early developmental events, has a low success rate and results from the artificial activation of oocytes, which may not include nuclear maturation. In Sminthopsis macroura, 24-h culture of advanced antral follicles in medium containing 10 μg mL^-1 porcine pituitary luteinising hormone (LH) yielded 60% of mature polarised oocytes with the first polar body; follicles cultured in medium without LH yielded only immature oocytes. Parthenogenetic activation of follicular, oviducal or uterine oocytes occurred when a two-step protocol was used. Sixty-one oocytes, exposed to 10 μm calcium ionophore A23187 for 10 min followed by 10 μg mL^-1 cycloheximide (protein synthesis inhibitor) for 5 h and then cultured for 20–24 h, were scored for signs of activation, namely extrusion of the second polar body and formation of the pronucleus. In each of 43 oocytes (70%), the extruded second polar body was present. Sixteen oocytes were analysed on slides after fixation and staining and, in 13 oocytes (81%) in this group, the female pronucleus was visible. No activation occurred following incubation of oocytes in medium containing Sr²⁺ for 5 h (n = 14), 8% ethyl alcohol solution for 8 or 12 min (n = 13) or 10 μm calcium ionophore A23187 (n = 13) for 10–20 min followed by culture for 20–24 h.