We have expressed the rat brain Ca2+/calmodulin (CaM)-dependent protein kinase type IV in insect cells. The recombinant enzyme is produced as a single polypeptide that migrates on SDS-polyacrylamide gel electrophoresis at 61 kDa. Recombinant CaM kinase IV undergoes slow CaM-dependent autophosphorylation. The autophosphorylation of CaM kinase IV occurs on serine residues but is not accompanied by the generation of a CaM-independent activity, as previously reported for the cerebellar enzyme. Comparison of peptide and protein phosphorylation by the recombinant CaM kinase IV and the cerebellar enzyme showed differences in their catalytic activities. The deduced primary sequence of CaM kinase IV contained a domain, 315Phe-Asn-Ala-Arg-Arg-Lys-Leu-Lys323, also found in the regulatory domain of CaM kinase II alpha (residues 293-300). Truncation of CaM kinase IV at Leu313 (at a position analogous to Leu290 in CaM kinase II alpha) generated a fully active, CaM-independent enzyme. This truncated enzyme no longer bound CaM. These data confirm that CaM kinase IV demonstrates intrasteric regulation by an autoinhibitory domain and provides insight into a potentially common mechanism for the regulation of the CaM-dependent multifunctional protein kinases. A number of synthetic peptides were examined for their phosphorylation by both CaM kinase II and IV. These studies showed that several peptides derived from phospholamban were preferential substrates for CaM kinase II whereas a peptide derived from S6 ribosomal protein was selectively phosphorylated by CaM kinase IV. Kinetic analysis of several peptide substrates suggests that while both CaM kinase II and IV recognize the sequence motif represented by R-X-X-T/S, other structural features are also involved in defining the unique substrate specificity of CaM kinase IV.