Background: Physical education (PE) can improve physical fitness; however, little research has evaluated PE's long-term influence. The purpose is to determine PE's longitudinal effects on fitness in a group of adolescent girls and to determine whether body mass index (BMI) status influenced any potential effects. Methods: Participants were enrolled in daily PE throughout their ninth grade. Data were collected at baseline and at the end of 9th, 10th, and 11th grades. BMI (kg/m[superscript 2]) was classified into Centers for Disease Control and Prevention percentiles. Fitness was estimated from a 3-stage step test (change in exercise heart rate (HR) indicated improved fitness) and analyzed using repeated measures general linear modeling with adjustments for baseline BMI, baseline age, ethnicity, intervention status, and moderate-to-vigorous physical activity outside PE. Results: Complete data were available for 131 girls (61% of sample) who were predominantly African American, 13.8 plus or minus 0.4 years, mean BMI 24.7 plus or minus 6.1 kg/m[superscript 2] at the ninth-grade baseline. Overall, stage 1 HR (bpm) decreased from baseline to 9th grade (-5.5 plus or minus 1.3, p less than 0.0001), baseline to 10th grade (-7.2 plus or minus 1.4, p less than 0.0001), and baseline to 11th grade (-7.0 plus or minus 1.5, p less than 0.0001). Change differed by BMI status; fitness improvement was significant for normal and overweight, but not obese girls. After 2 years, change in stage 1 HR (bpm) was significantly different between BMI categories: normal-weight girls versus obese girls (-10.0 plus or minus 3.2, p = 0.002) and overweight girls versus obese girls (-9.3 plus or minus 3.9, p = 0.02). Conclusion: Fitness differences by BMI exist after a PE program in adolescent girls: fitness improved and was maintained in normal-weight and overweight girls, whereby obese girls had no improvement in fitness. (Contains 4 figures and 1 table.)