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Sexual segregation, in which males and females form separate groups for most of the year, is common in sexually dimorphic ungulates. We tested multiple hypotheses to explain sexual segregation in bison (Bison bison) at National Bison Range, Montana and Fort Niobrara National Wildlife Refuge, Nebraska during June-August of 2002-2003. Fieldwork involved use of GPS to record space use by segregated groups, vegetation transects to measure forage availability, fecal analyses to document diet composition and quality, and behavioural observations to characterize activity budgets. During sexual segregation, males in bull groups used areas with greater per capita abundance of forage, higher proportion of weeds, and less nutritious grasses (as indicated by lower % fecal nitrogen) compared with females in cow or mixed groups. However, there was no difference between the sexes in activity budgets, predation risk factors, or distance to water. Single-sex bull groups were no more synchronized in activity than mixed groups. These results support the ’sexual dimorphism-body size hypothesis’, which proposes that males segregate from females because their larger body size requires more abundant forage, while longer ruminal retention permits efficient use of lower-quality forage. The gastrocentric model, based on the digestive physiology and foraging requirements of dimorphic ungulates, supplies the most likely proximate mechanism for bison sexual segregation. Our results would also partly support the ’reproductive strategy-predation risk hypothesis’ if females form large groups to reduce predation risk. The predictions of the ’activity budget hypothesis’ were not supported for bison.