BACKGROUND: To measure hamstring flexibility, the active knee extension (AKE) test is preferred over the straight leg raise (SLR) test as it can be used to measure hamstring flexibility more selectively. However, hamstring stretching is primarily conducted in the SLR position (maximal hip flexion in the supine position) as it allows for maximal hip flexion in the supine position. OBJECTIVE: This study evaluates the effects of proprioceptive neuromuscular facilitation (PNF) stretching in the AKE position (maximal knee extension with 90 degrees flexion of the hip in the supine position) on hip, knee, and ankle flexibility. METHODS: SLR, AKE, and active dorsiflexion (ADF) tests were used to determine the range of motion (ROM) before (pre-ROM) and after (post-ROM) stretching. PNF stretching consisted of maximal isometric knee flexion at the end range with external resistance to prevent knee flexion. One set of PNF stretches (five trials of six seconds each) was conducted. RESULTS: The post-ROMs of hip, knee, and ankle measured via the SLR, AKE, and ADF tests, respectively, were significantly higher than the pre-ROMs. CONCLUSIONS: The improvement in knee flexibility was greater than the improvement in hip and ankle flexibility. The AKE position is recommended in clinical settings during PNF stretching for individuals with hamstring tightness. Furthermore, PNF stretching in the AKE position increases the ADF ROM.