Our goal was to assess cross-sectional and longitudinal associations between physical activity and sedentary behaviors (television viewing, computer and video-game usage, and reading) with health-related QoL. Of 2353 children surveyed (median age: 12.7 years), 1216 were resurveyed 5 years later, and 475 were newly recruited into the study (N = 1691). Children completed detailed activity questionnaires. Health-related QoL was assessed by using the Pediatric Quality of Life Inventory (PedsQL). Cross-sectionally, after multivariable adjustment, adolescents in the highest versus lowest tertile of time spent in outdoor physical activity and television viewing had a higher (P(trend) = .001) and lower (P(trend) = .0003) total PedsQL score, respectively. Adolescents who remained in the highest tertiles compared with those in the lowest tertiles of total physical activity over the 5 years had significantly higher scores in the following areas: total (P(trend) = .04), physical summary (P(trend) = .0001), and social (P(trend) = .02) domains. Conversely, those in the highest versus lowest tertile of screen-viewing time during follow-up reported significantly lower values in the following areas: total score (6.34-unit difference), physical summary (4.86-unit difference), psychosocial summary (7.09-unit difference), and emotional (8.33-unit difference) and school (9.78-unit difference) domains. Regular physical activity over the long-term was associated with higher perceived health-related QoL among adolescents. Conversely, lower PedsQL scores were observed among those who spent the most time in screen-viewing activities. Improved understanding of these relationships could help in developing interventions to promote general well-being among adolescents.