Blebbistatin was discovered in a screen for chemical inhibitors of nonmuscle myosin IIA (NMIIA) ATPase activity. It inhibits blebbing and cytokinesis in Xenopus and human cells. We tested its activity against a number of other conventional and unconventional myosin isoforms. Blebbistatin inhibited the actin-activated MgATPase activity of NMIIA heavy meromyosin (HMM), NMIIB (HMM), smooth muscle HMM, and several striated muscle HMMs. The amount of blebbistatin required for half maximal inhibition of the actin-activated MgATPase is ranged from 0.4 micromolar for rabbit skeletal muscle HMM to 80 micromolar for smooth muscle HMM. Blebbistatin, even at 100 micromolar, did not effectively inhibit the activity of rat myo1b, Acanthamoeba myosin IC, myosin-V and myoxin-X. Blebbistatin completely inhibited the movement of rhodamine-phalloidin labeled F-actin by NMIIA and skeletal muscle HMM. This effect was reversible upon wash out of blebbistatin. The inhibitory activity of blebbistatin was destroyed by illumination with blue light (488 nm). This property could be useful in studying the reversibility of blebbistatin inhibition of contractile processes in cells and for synchronizing contractile events, but care must be taken since the inactivation of blebbistatin appears to be associated with the release of free radicals. We have characterized the kinetic mechanism for the blebbistatin inhibition of myosin. The main step that is affected in the release of phosphate which means that the steady-state product in the presence of blebbistatin will bind weakly to actin. We have characterized the inhibitory properties of several derivatives of blebbistatin in hopes of finding a derivative with higher affinity or one that does not inactivate in the presence of blue light. In addition, we are searching for molecules that will discriminate between the various nonmuscle myosin II isoforms. The preliminary data suggests that discrimination may be possible, but so far the affinity of the derivatives are weaker than that of the original blebbistatin. A nitro derivative has been prepared that does not photoinactivate.