A new class of stimulators of basal activity of a number of calmodulin-dependent enzymes have been previously isolated from bovine hypothalamus. One of these stimulators, denoted as C3, has been purified to homogeneity by reverse phase HPLC and tentatively identified as thymosin beta 4 (1-39) by mass spectrometry and Edman microsequence analysis. The stimulating effect of C3 on rabbit skeletal muscle MLCK basal activity was compared with that of thymosin alpha 1 and thymosin beta 4 (16-38). Evidence is presented that all the indicated compounds are Ca(2+)-independent high-affinity MLCK stimulators. The potency of the stimulators in activating the enzyme was: C3 greater than beta 4 greater than (CaM+Ca2+) greater than alpha 1.