The purpose of this study was to develop and apply a general method for estimating the architectural properties of human muscles in vivo. The method consists of a two-phase, nested optimization procedure in which the values of peak isometric force, optimal muscle-fiber length, and tendon slack length are calculated for each musculotendon actuator, knowing muscle volume and the minimum and maximum physiological lengths of the actuator. In phase I, the positions of the bones and the activation levels of the muscles are found by maximizing the isometric torque developed for each degree of freedom at each joint. In phase II, the architectural properties of each musculotendon actuator are found by matching the strength profile of the model to that measured for subjects. The method is used to estimate the architectural properties of 26 major muscle groups crossing the shoulder, elbow, and wrist. Wherever possible, the model calculations are compared against measurements obtained from anatomical studies reported in the literature. Architectural data obtained from our work should be useful to researchers interested in developing musculoskeletal models of the upper limb.