The interactive influence of preschool children's level of physical activity, sex, and time on the degree of sex segregation was assessed. A sample of nursery school children was observed across much of a school year, and levels of physical activity and sex segregation were sampled during their free play periods. Following sexual selection theory, we predicted a Sex × Time × Physical Activity interaction on segregation such that high-activity girls early in the school year would interact with boys but, with time, the high-activity girls would be segregated among themselves. Boys (both high- and low-activity) should remain segregated across the year. The hypothesis was supported, and results are discussed in terms of the interactive role of biology and socialization on sex segregation.