Since the proximal tendon of the gastrocnemius muscle wraps around the posterior aspect of the tibia, its contraction could potentially strain the anterior cruciate ligament (ACL) by pushing the tibia anteriorly. However, the relationship between contraction of the gastrocnemius muscle and ACL strain has not been studied in vivo. The objectives of this study were to evaluate the ACL strain response due to isolated contractions of the gastrocnemius muscle and to determine how these strains are affected by cocontraction with the hamstrings and quadriceps muscles. Six subjects with normal ACLs participated in the study; they underwent spinal anesthesia to ensure that their leg musculature was relaxed. Transcutaneous electrical muscle stimulation (TEMS) was used to induce contractions of the gastrocnemius, quadriceps and hamstrings muscles while the strains in the anteromedial bundle of the ACL were measured using a differential variable reluctance transducer. The ACL strain values produced by contraction of the gastrocnemius muscle were dependent on the magnitude of the ankle torque and knee flexion angle. Strains of 2.8% and 3.5% were produced at 5 degrees and 15 degrees of knee flexion, respectively. The ACL was not strained at 30 degrees and 45 degrees. Changes in ankle angle did not significantly affect these strain values. Co-contraction of the gastrocnemius and quadriceps muscles produced ACL strain values that were greater than those produced by isolated activation of either muscle group when the knee was at 15 degrees and 30 degrees. Co-contraction of the gastrocnemius and hamstrings muscles produced strains that were higher than those produced by the isolated contraction of the hamstrings muscles. At 15 degrees and 30 degrees of knee flexion. the co-contraction strain values were less than those produced by stimulation of the gastrocnemius muscle alone. This study verified that the gastrocnemius muscle is an antagonist of the ACL. Since the gastrocnemius is a flexor of the knee, this finding may have important clinical ramifications in ACL rehabilitation since flexor torques are generally thought to be protective of a healing ACL graft.