The effects of gonadectomy and hormone treatment on spatial learning were evaluated in adult male and female rats using a modified version of a 12-arm radial maze task. In this version, procedures were used to minimize the effectiveness of strategies less reliant on working and reference memory. Results demonstrate significant sex differences favoring male performance on the working memory component of the task. In contrast, females performed slightly better than males on the reference memory component of the task. In females, ovariectomy produced a decrease in overall accuracy (i.e., an increase in the number of arm entries necessary to obtain all food pellets), as well as declines in working and reference memory performance. Both accuracy and working memory performance, but not reference memory performance, were restored by estradiol treatment. In males, castration impaired working memory performance, but did not significantly affect overall accuracy or reference memory performance. Surprisingly, all groups of males performed poorly on the reference memory component of the task, and testosterone treatment appeared to worsen, rather than improve, both accuracy and reference memory performance in males. This may reflect a male preference for certain strategies which were rendered ineffective on this task. Significant sex differences, as well as treatment effects, on arm preference patterns were also detected; however, these differences were not sufficient to account for the effects of sex and treatment on acquisition. Collectively, the data demonstrate robust effects of gonadectomy and hormone treatment on acquisition of this modified radial arm maze task in females, with lesser effects in males.