In this paper a new navigation system, KIN-Nav, developed for research and used during 80 anterior cruciate ligament (ACL) reconstructions is described. KIN-Nav is a user-friendly navigation system for flexible intraoperative acquisitions of anatomical and kinematic data, suitable for validation of biomechanical hypotheses. It performs real-time quantitative evaluation of antero-posterior, internal-external, and varus-valgus knee laxity at any degree of flexion and provides a new interface for this task, suitable also for comparison of pre-operative and post-operative knee laxity and surgical documentation. In this paper the concept and features of KIN-Nav, which represents a new approach to navigation and allows the investigation of new quantitative measurements in ACL reconstruction, are described. Two clinical studies are reported, as examples of clinical potentiality and correct use of this methodology. In this paper a preliminary analysis of KIN-Nav's reliability and clinical efficacy, performed during blinded repeated measures by three independent examiners, is also given. This analysis is the first assessment of the potential of navigation systems for evaluating knee kinematics.