Smooth muscle and nonmuscle myosin II are regulated by phosphorylation of the 20 kDa regulatory light chain (RLC) located in the neck region. This region contains a single alpha-helical segment of the myosin heavy chain and the RLC and the essential light chain (ELC). There are two vertebrate genes encoding for nonmuscle myosin IIs, termed IIA and IIB. Platelets and lymphocytes express only the IIA isoform, whereas brain expressed predominantly the IIB isoform. Most other cell express nearly equal ratios of IIA and IIB where they are differentially localized. Previous work in our lab has revealed that nonmuscle myosin IIA and myosin IIB have differing enzymatic and motile properties. In order to better understand the basis for this difference, we have engineered myosin IIA and myosin IIB S-1 like fragments for baculviral expression. Preliminary characterization of the steady state actin-activated MgATPase reveals that IIA S1 has a higher enzymatic rate than that of IIB S1. Both proteins express well and should produce sufficient material for transient kinetic analysis and for collaborative experiments on the single molecule mechanics. - nonmuscle myosin; regulatory domain; phosphorylation; baculoviral expression; MgATPase