The kinase responsible for phosphorylating human platelet myosin light chains has now been isolated in a Ca2 ion-dependent form. It had previously been isolated in a Ca2 ion-independent form (Daniels and Adelstein, Biochem. 15:2370, 1976). The Ca2 ion-dependent kinase was purified using CDR-affinity chromatography, indicating that the 16,500 Ca2 ion-dependent regulatory protein (CDR), known to be present in platelets as well as most other nonmuscle and muscle cells, plays an important role in regulating the kinase. The partially purified kinase is inactive in the absence of CDR and required both Ca2 ion and CDR for full activity.