Regulatory domain elements of the non-calcium-dependent protein kinase Cdelta (nPKCdelta), including either or both of the cysteine-rich regions Cys1(delta) and Cys2(delta), were expressed as fusion proteins with glutathione-S-transferase and characterized using liposomal or mixed micellar phorbol ester binding assays. Fusion proteins containing Cys2(delta) bound phorbol-12,13-dibutyrate (PDBu) efficiently in the assay employing phosphatidylserine (PS) vesicles, while no significant binding was seen for proteins containing only Cys1(delta). Likewise, in mixed micellar assays, fusion proteins with Cys2(delta) bound PDBu with high affinity (Kd: 14-37 nM) and to significant stoichiometric levels (0.23-0.66 mol/mol), but no binding could be detected for proteins with Cys1(delta) only. The PS dependence of PDBu binding to Cys2(delta) was highly cooperative with Hill numbers lying in the range of 2.5-5.2. These results demonstrate the presence of striking functional differences between the cysteine-rich regions of nPKCdelta and the calcium-dependent isoform, cPKCgamma, where both cysteine-rich regions represent functional PDBu binding elements.