Rho-kinase II (ROCK-II) is a serine/threonine kinase that is involved in regulation of smooth muscle contraction and has been shown to contribute to the early stages of axon formation in neurons and the regulation of the neuronal cytoskeleton. Much of what is known about Rho-kinase function comes from cell-biological studies, whereas a paucity of biochemical characterization exists for the enzyme. In an effort to characterize ROCK-II biochemically we have cloned a truncated form of human ROCK-II comprising amino acids 1-543 and overexpressed it in Sf-21 cells. Utilizing the Sf-21/baculovirus expression system we isolated milligram quantities of ROCK-II (1-543) and purified the enzyme to near homogeneity. Optimal expression conditions revealed that infection of Sf-21 cells at a multiplicity of infection of 10 for 72h yielded maximal protein expression. Expression of ROCK-II (1-543) as an N-terminal Flag fusion protein allowed a single-step purification yielding greater than 90% homogeneous protein as assessed by SDS-PAGE. Enzyme activity was linear over a range of enzyme concentrations and times. Capture of phosphorylated, biotinylated peptides on streptavidin membrane allowed assessment of peptide substrate preference and measurement of steady-state rate constants. The data indicated that an 11-mer peptide containing Ser235/Ser236 of the S6 ribosomal protein and a 12-mer peptide containing Thr508 of LIM kinase were preferred substrates for ROCK-II (1-543). Finally, staurosporine had an IC(50) value 215-fold more potent than that of the ROCK inhibitor Y-27632. Collectively these data lay the foundation for the beginning of a biochemical characterization for this enzyme and provide methodology for more detailed biochemical, biophysical, and kinetic analysis.