The goal of this study was to analyze the architecture of the cat biceps femoris (BF), a multifunctional hamstring muscle, and to evaluate the relationships between muscle architecture, limb position, and muscle function during natural movement. The BF muscle consists of three neuromuscular compartments: anterior (BFa), middle (BFm) and posterior (BFp). Each compartment is innervated by a separate nerve branch. Nerve branch stimulation and 2-dimensional surface EMG recordings showed that individual compartment territories were discrete and non-overlapping with well-defined borders. Comparisons of the three compartments revealed consistent differences in architecture, relationship to the skeleton, and function. The BFa crossed only the hip joint and appears to function as a pure hip extensor. The BFm had equal lever arm lengths to the hip and knee joints, appears to function as a hip extensor, and may contribute to knee flexion or femoral rotation. The BFp had a greater lever arm to the knee, functions as a knee flexor, and may contribute to hip extension, femoral rotation or ankle extension. Measurements of individual fascicles from the three compartments revealed a surprising range of lengths, 3.3-12.0 cm. Microdissection of gold-stained tissue showed that fascicles from all compartments were comprised of interdigitated, short fibers (range: 0.6-5.0 cm; average 2.14 cm) arranged in-series in fascicles, running parallel to the origin-insertion axis of each muscle compartment. In regions of fiber interdigitation, the fiber endings were round and tapered (taper lengths: 1-11 mm) although flat, tapering endings like ribbons were occasionally found. As hip and knee joint angles were varied over physiological ranges corresponding to minimal to maximal muscle length, fascicles of the three compartments changed length disproportionately. Long BFa fascicles maximally lengthened 10-18%, consistent with in vivo length measures during treadmill locomotion. However, the long BFp fascicles lengthened 25-45%, and the relatively short fascicles near the BFm/BFp border maximally lengthened 45-53%. How do these unexpectedly large length changes affect sarcomere lengths? Using laser diffraction to measure sarcomeres, static fascicle and sarcomere lengths were compared in muscles that went into rigor mortis after fixing the hip and knee joint angles. Sarcomeres within the short BFm/BFp and long BFp fascicles consistently lengthened proportionately less than the whole fascicle. It remains to be determined how and where the fascicle length changes are dissipated in the connective tissue between the interdigitated muscle fibers and whether such a series-compliance operates during the large excursions over which this muscle normally works.