A light chain of 18 000 daltons of native actomyosin isolated from rabbit skeletal muscle was removed by DTNB-treatment. Investigated were the differences in superprecipitation and ATPase activity of LC2-deficient and control actomyosin. The superprecipitation of control actomyosin develops in two phases, with high amplitude and kinetics dependent on the Ca++ concentration. On the other hand, superprecipitation of treated actomyosin develops in a single phase, with low amplitude and a kinetics independent of the Ca++ concentration. The partial lack of LC2 leads to the loss of Ca++-sensitivity of ATPase activity. On the basis of the results, LC2 has been assumed to fix that conformation state of myosin heads which is required for the functioning of the troponin system on the one hand, and a cooperation between myosin heads on the other. The regulatory function of LC2 is manifest in controlling the actin-myosin-ATP interaction, at the activated state of the troponin system, in an extent depending on the actual Ca++ concentration.