A method has been developed for measuring the level of phosphorylation of myosin regulatory light chains (MLC2) by the endogenous myosin light chain kinase in mechanically skinned skeletal muscle fibres. The method was used to characterize the endogeous MLC2 phosphorylation capacity of single fast-twitch fibres from the rat and to investigate the relationship between the endogenous MLC2 phosphorylation and the Ca(2+)-activated force. The results show that (1) about 50% of MLC2 were 32P-phosphorylated after activation of the skinned fibre preparation by 30 microM [Ca2+] for longer than 30 s, but that there was variability between fibres; (2) most of the endogenous phosphorylating system diffused out of the skinned fibre preparation after 5 min exposure to an aqueous solution; (3) the MLC2 phosphorylation by the endogenous phosphorylating system followed with a delay of the order of 1-2 s after the sudden rise in [Ca2+] from below 10 nM to 30 microM; and (4) the sensitivity of the contractile apparatus to Ca2+ was markedly increased when the MLC2 were phosphorylated by the endogenous phosphorylating system following a rise in [Ca2+]. The Kd for MgATP of the endogenous MLC2 phosphorylating system was estimated to be less than 300 microM. These results unequivocally demonstrate that prolonged activation of the fast-twitch muscle fibre leads to increased Ca2+ sensitivity of the contractile apparatus and that mechanically skinned fibres can be successfully used to study the regulation of the endogenous MLC2 phosphorylation capacity at single muscle fibre level.