Phosphorylation of Ca2+/calmodulin-dependent protein kinase I (CaM KI) at Thr-177 by recombinant rat Ca2+/calmodulin-dependent kinase kinase B (CaM KKB) modulates the kinetics of synapsin-(4-13) peptide phosphorylation by reducing the Km 44-fold and decreasing the KCaM 4-fold. There is also a slight decrease in Km for ATP and increase in enzyme Vmax. A synthetic peptide substrate from the yeast transcription factor, ADR1-(222-234)G233 is a 15-fold better substrate for the Thr-177 dephospho-form of CaM KI than synapsin-(4-13). The Thr-177 dephospho-enzyme has a Km and Vmax for ADR1-(222-234)G233 similar to the values with synapsin-(4-13) using the Thr-177 phosphorylated enzyme. Likewise, with ADR1-(222-234)G233 as substrate, phosphorylation of Thr-177 or substitution of T177A had very little effect on the kinetic values. Using chimeric peptides between synapsin-(4-13) and ADR1-(222-234)G233 we found that N-terminal basic residues at P-7 and P-6 positions were sufficient to allow efficient phosphorylation by the Thr-177 dephospho-form of CaM KI. Phosphorylation of Thr-177 expands the substrate specificity of CaM KI and is not merely an "on-off" switch for kinase activity.