Synthetic peptides corresponding to the phosphorylation site in the myosin regulatory light chain from smooth muscle, LysLysArgAlaAlaArgAlaThr SerAsnValPheAla ([Ala14,15]MLC(11-23)) and containing a variety of hydroxyamino acid analogs at position 19, were tested as substrates for the smooth muscle myosin light chain kinase. Peptide analogs containing either d-serine or cis-hydroxyproline were not phosphorylated. The corresponding trans-hydroxyproline containing peptide was poorly phosphorylated with a Km of 2.3 μm and a Vmax of 3 × 10-3 μmol · min-1 · mg-1 compared to a Km of 12.5 μm and a Vmax of 1.43 μmol · min-1 · mg-1 for the parent peptide. All three hydroxyamino acid analog peptides acted as relatively potent inhibitors of myosin light chain phosphorylation with Ki values in the range 7.5-10 μm, comparable to 7 μm for the parent peptide. Thus the failure of the hydroxyamino acid analog peptides to act as effective substrates was not the result of poor binding to the enzyme. In contrast, the same substitutions made in the peptide substrate for the cAMP-dependent protein kinase resulted in poor inhibitors. It is likely that the hydroxyl group of the substituting amino acids in the myosin light chain peptide analogs is not presented in the correct orientation in the active site for transfer of the phosphate group.