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Multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) is a mediator of calcium signals in diverse signaling pathways. In human lymphocytes and epithelial tissues, CaM kinase activates a chloride channel via a Ca(2+)-dependent pathway which is preserved in cystic fibrosis. To characterize the CaM kinase present in these tissues we have cloned an isoform of this kinase from human T lymphocytes. We show the cDNA structure of two variants of this human CaM kinase, gamma B and gamma C, which are predicted to translate to 518 and 495 amino acids, respectively. Amino acid differences between these isoforms and the rat brain gamma isoform (which we refer to as gamma A) are localized to the variable domain. We used RNase protection of this variable region to reveal the level of expression of gamma B and gamma C CaM kinase mRNAs in nine human tissues and cell lines. When transfected into Jurkat T cells, the gamma B cDNA encoded a functional kinase which cosedimented on sucrose gradients with endogenous T cell CaM kinase activity and formed a large multimeric enzyme. The recombinant gamma B isoform displayed two phases of autophosphorylation characteristic of CaM kinases, including the phase which converts it to a partially Ca(2+)-independent species. Site-directed mutagenesis of the predicted autoinhibitory domain yielded a mutant which was approximately 37% active in the absence of Ca2+/calmodulin, confirming the region as critical for autoregulation, and suggesting this mutant as a tool for studying the role of CaM kinase in nonneuronal tissues.