Protein kinase C (PKC) is a key enzyme family involved in cellular signal transduction. The binding of endogenous diacyl glycerol (DAG) to the cysteine-rich domain (CRD) of PKC is associated with normal cell signaling and function. In contrast, the binding of exogenous phorbol esters to the CRD of PKC is considered to be a key initiating event in tumor promotion. Conventional PKC isozymes (PKC alpha, beta I, beta II, and gamma) contain two CRDs, both of which are candidates for the phorbol ester binding site. In order to elucidate the binding requirements of phorbol esters and to obtain information on the phorbol ester binding site in native PKC gamma, several key chemical characteristics of the first and the second CRDs consisting of ca. 50 amino acids of rat PKC gamma (gamma-CRD1 and gamma-CRD2) were examined. In the presence of Zn2+ and phosphatidylserine (PS), both CRDs gave similar Kd values (65.3 nM for gamma-CRD1, 44.1 nM for gamma-CRD2) in phorbol 12,13-dibutyrate (PDBu) binding assays. In comparison, the binding affinity of PDBu for native rat PKC gamma was found to be 6.8 nM. Zn2+ was shown to play an important role in the folding and PDBu binding of both CRDs. A Zn(2+)-induced conformational change was observed for the first time by CD spectroscopic analysis of the complexed and uncomplexed CRDs. Relative to the pronounced Zn2+ effect, most divalent first row transition metal ions along with Ca2+, Mg2+, and Al3+ were ineffective in folding either CRD. Notably, however, Co2+ exhibited a gamma-CRD1-selective effect, suggesting that metal ions, not unlike extensively used organic probes, might also become effective tools for controlling isozyme selective activation of PKC. Moreover, group Ib (Cu2+ and Ag+) and group IIb element ions other than Zn2+ (Cd2+ and Hg2+) were found to abolish PDBu binding of both CRDs. Importantly, these inhibitory effects of Cu2+, Ag+, and Cd2+, and Hg2+ were also observed with native PKC gamma. These results indicate that recent reports on the modulation of conventional PKC by heavy metal ions could be explained by their coordination to the CRDs. While the similar affinities of gamma-CRD1 and gamma-CRD2 for PDBu suggest that either site qualifies as the PDBu binding site, new molecular probes of these CRD3 have now been identified that provide information on the preferred site. These novel ligands (5a and 5b) were synthesized by aza-Claisen rearrangement of (-)-N13-desmethyl-N13-allylindolactam-G (4). These compounds did not significantly affect the specific PDBu binding of gamma-CRD1 but did inhibit that of gamma-CRD2 with similar potency to (-)-indolactam-V. Moreover, these new probes did not significantly inhibit the PDBu binding of native PKC gamma. (-)-Indolactam-V itself bound almost equally to gamma-CRD1, gamma-CRD2, and native PKC gamma. These results suggest that the major PDBu binding site in native PKC gamma is the first CRD, not the second CRD, unlike the novel PKCs.