Background: This study examined differences in students' time spent in physical activity (PA) across secondary schools in rural, suburban, and urban environments and identified the environment-level factors associated with these between school differences in students' PA. Methods: Multilevel linear regression analyses were used to examine the environment- and student-level characteristics associated with time spent in PA among grades 9 to 12 students attending 76 secondary schools in Ontario, Canada, as part of the SHAPES-Ontario study. This approach was first conducted with the full data set testing for interactions between environment-level factors and school location. Then, school-location specific regression models were run separately. Results: Statistically signifiant between-school variation was identified among students attending urban (sigma[superscript 2] [subscript [mu]o] = 8959.63 [372.46], suburban (sigma[superscript 2] [subscript [mu]o] = 8918.75 [186.20]), and rural ((sigma[superscript 2] [subscript [mu]o] = 9403.17 [203.69]) schools, where school-level differences accounted for 4.0%, 2.0%, and 2.1% of the variability in students' time spent in PA, respectively. Students attending an urban or suburban school that provided another room for PA or was located within close proximity to a shopping mall or fast food outlet spent more time in PA. Conclusion: Students' time spent in PA varies by school location and some features of the school environment have a different impact on students' time spent in PA by school location. Developing a better understanding of the environment-level characteristics associated with students' time spent in PA by school location may help public health and planning experts to tailor school programs and policies to the needs of students in different locations. (Contains 5 tables.)