Telomerase, a specialized ribonucleoprotein reverse transcriptase that directs the synthesis of telomeric DNA, is repressed in normal human somatic cells, but is activated in most cancers. Little is known concerning how telomerase activity is activated and maintained in cancer cells. We have shown previously that inhibition of protein kinase C (PKC) decreases the telomerase activity of human nasopharyngeal carcinoma (NPC) cells. Here, we provide evidence that the decrease of telomerase activity by PKC inhibition is not mediated by transcriptional down-regulation of hTERT, the catalytic protein of human telomerase. In vitro phosphorylation studies revealed that exogenous addition of PKC-alpha, -betaI, -delta or -zeta led to restoration of telomerase activity in the crude extracts of PKC-inhibited NPC cells. However, depletion of PKC-alpha and -betaI in vivo had no detectable effect on the telomerase activity of NPC cells. Using antisense oligonucleotides against individual PKC isotypes, we observed that telomerase activity was inhibited only by the antisense oligonucleotide against PKC-zeta but not by those against PKC-alpha, -betaI or -delta. Taken together, these data demonstrate that PKC participates in the regulation of telomerase activity by direct or indirect phosphorylation of telomerase proteins, and that PKC-zeta is the PKC isotype that functions in vivo in the NPC cells.