This study was designed to determine the in situ strains, stresses, and loads in the medial collateral ligament (MCL) of skeletally immature and mature rabbits. Using a noncontact method, the magnitudes of the in situ strains were first determined as a function of knee flexion angle. The MCL was divided into three anatomical regions (anterior, middle, and posterior) across its width. For strain measurements, the variation of a gauge length in these regions was obtained in the intact knee at 60, 90, and 120 deg of flexion. Subsequently, all soft tissues around the knee were dissected away, leaving the femur-MCL-tibia (FMT) complex. The MCL was allowed to retract freely and the new length, called the zero length, was measured. From this, the in situ strains were determined. To obtain the stress-strain relationship of the FMT complex, the specimens were subjected to tensile testing. Knowing the in situ strains and the stress-strain relationship, the in situ stresses in the three anatomical regions of the MCL were determined as a function of knee flexion angle. Multiplying these stresses by 1/3 of the cross-sectional area and summing the loads thus calculated, the in situ loads of the MCL were obtained. Our data suggest that the in situ load in the MCL is not large within the range of knee flexion angles studied, i.e., 1.4 to 2.7 N for the skeletally immature animals and 3.0 to 5.8 N for the skeletally mature animals. An increase in the in situ load with skeletal maturation was demonstrated.