Fast solution switching techniques in single myofibrils offer the opportunity to dissect and directly examine the sarcomeric mechanisms responsible for force generation and relaxation. The feasibility of this approach is tested here in human cardiac myofibrils isolated from small samples of atrial and ventricular tissue. At sarcomere lengths between 2.0 and 2.3 μm, resting tensions were significantly higher in ventricular than in atrial myofibrils. The rate constant of active tension generation after maximal Ca²⁺ activation (k _ACT) was markedly faster in atrial than in ventricular myofibrils. In both myofibril types k _ACT was the same as the rate of tension redevelopment after mechanical perturbations and decreased significantly by decreasing [Ca²⁺] in the activating solution. Upon sudden Ca²⁺ removal, active tension fully relaxed. Relaxation kinetics were (1) much faster in atrial than in ventricular myofibrils, (2) unaffected by bepridil, a drug that increases the affinity of troponin for Ca²⁺, and (3) strongly accelerated by small increases in inorganic phosphate concentration. The results indicate that myofibril tension activation and relaxation rates reflect apparent cross-bridge kinetics and their Ca²⁺ regulation rather than the rates at which thin filaments are switched on or off by Ca²⁺ binding or removal. Myofibrils from human hearts retain intact mechanisms for contraction regulation and tension generation and represent a viable experimental model to investigate function and dysfunction of human cardiac sarcomeres.