The ovary of the brushtail possum (Trichosurus vulpecula) secretes steroids; however, little is known about the identity of the steroidogenic cells in the ovary. The aim of the present study was to determine the identity of the ovarian cell types expressing mRNAs encoding proteins important for steroidogenesis and determine at what stage of follicular development they are expressed. The genes examined were those for steroidogenic factor-1 (SF-1), steroidogenic acute regulatory protein (StAR), cytochrome P450 side chain cleavage (P450_scc), 3β-hydroxysteroid dehydrogenase/Δ⁵,Δ⁴ isomerase (3βHSD), cytochrome P45017αhydroxylase (P45017αOH), and P450 aromatase (P450_arom). None of the genes examined were expressed in oocytes at any stage of follicular development. SF-1 was expressed in granulosa cells from the type 2 or the primary stage of development and thereafter to the preovulatory stage. In addition, the theca interna of small and medium-size antral but not preovulatory follicles and the interstitial glands and corpora lutea expressed SF-1 mRNA. Granulosa cells of preantral and small to medium-size antral follicles were not capable of synthesizing steroids from cholesterol because they did not contain P450_scc mRNA. However, granulosa cells of many of the small to medium-size antral follicles expressed P450_arom and 3βHSD mRNA. The interstitial glands, theca interna, and corpus luteum expressed StAR, P450_scc, 3βHSD, and P45017αOH mRNA, suggesting that these tissues are capable of synthesizing progestins and androgens. The corpus luteum expressed P450_arom, indicating that this tissue also has the potential to secrete estrogens in this species.