This study investigated the reproductive biology of a captive population of brush-tailed phascogales Phascogale tapoatafa. A ‘round robin’ mating system was established in which seven adult male animals were rotated through seven adult females. Changes in vaginal cytology and peripheral plasma progesterone and oestradiol levels were monitored to determine features of the female reproductive cycle. Regular measurement of scrotal diameter, collection of testicular biopsies and monitoring of plasma testosterone levels was used to assess male reproductive activity. Oestrous cycle length was estimated as 40 ± 5 days (n = 7) and gestation length as 27 ± 5 days (n = 7). Plasma progesterone levels showed no significant variation. This negative result is attributed to the small sample size (n = 4). Plasma oestradiol levels however, followed the predicted pattern, with an increase in peripheral concentration occurring prior to the breeding season. Oestradiol concentration peaked at 246.9 ± 169.2 pg ml−1 (mean ± SE) just before mating and declined to 24.3 ± 12.0 pg ml−1 after mating. Histological studies of testicular biopsies revealed that spermatogenesis does not cease until after the breeding season has begun, in contrast to Antechinus species, in which spermatogenesis ceases before breeding. Plasma testosterone levels increased at the onset of the breeding season, peaking at 6.3 ± 0.60 ng ml−1. Testosterone concentrations remained high until approximately 3 weeks after breeding, followed by a decline to pre-breeding levels. Litter size and sex ratio did not vary significantly between first- and second-year females. Females did not undergo a second oestrous cycle when pouch young were removed 15 and 17 days after parturition.