A protein kinase C-related cDNA encodes a novel phorbol ester receptor/protein kinase, nPKCε, clearly distinct from the four “conventional” PKCs Ohno, S., Akita, Y., Konno, Y., Imajoh, S., & Suzuki, K. (1988) Cell 53, 731–741. We purified nPKCε from COS cells transfected with nPKC cDNA and compared its enzymatic properties with a conventional PKC, PKCa. nPKCε was eluted from a hydroxyapatite column at a position coincident with type II PKC and thus was separated from type III PKC (PKCa), the only PKC expressed in COS cells. The protein kinase activity of nPKCε is activated by phospholipids and diacylglycerols (or phorbol esters) in a manner similar to conventional PKCs. However, the cofactor dependencies and substrate specificites were clearly different from PKCα. A phospholipid, cardiolipin, enhances the kinase activity three- to fourfold compared with phosphatidylserine. The optimum Mg2+ concentration (3 mM) is clearly different from those of conventional PKCs (10–20 mM). The activation of nPKCε by these cofactors is totally independent of Ca2+. Similar to conventional PKCs, nPKCe autophosphorylates serine and threonine residues, indicating the specificity of the kinase to these amino acid residues. However, it shows a clearly different substrate specificity against exogenous substrates in that myelin basic proteins rather than histone are good substrates. These properties of nPKCε permit clear discrimination of nPKCε from conventional PKCs.