The main regulatory mechanism of smooth muscle contraction involves Ca2+/calmodulin (CaM)-dependent phosphorylation of myosin (CDPM), by myosin light chain kinase (MLCK). It is also known that the increase in intracellular Ca2+ and phosphorylation of myosin occurs within a short time under physiological conditions, but the muscle tension may persist for a longer period of time. However, the mechanism of this phenomenon is still not clear. We hypothesize that MLCK also phosphorylates myosin in a Ca2+/CaM-independent manner (CIPM). The difference between CIPM and CDPM are as follows. Firstly, the extent of CIPM by MLCK was temperature-independent, whereas CDPM by MLCK was apparently decreasing with increasing temperature. Secondly, in contrast to the decreased extent of CDPM, the prolongation of incubation time did not decrease the extent of CIPM. Thirdly, a high concentration of K＋ influences CIPM less than CDPM. Furthermore, the MLCK inhibitor ML-9 significantly inhibited CDPM by MLCK but not CIPM by MLCK. Lastly, arachidonic acid selectively increased CIPM by MLCK but not CDPM by MLCK. Finally, the activity of Mg2+-ATPase of myosin followed the sequence as this: CDPM > CIPM > unphosphorylated myosin. Our results revealed some primary features of CIPM by MLCK.