With the amino acid sequences of all reported Akt kinase physiological substrates, the possible Akt kinase substrate specificity has been suggested. The serine/ threonine residue to be phosphorylated in these proteins is placed within stretches of amino acids with homology, and the arginine residues on the ؊5 and ؊3 positions and a hydrophobic amino acid on the ؉2 position are conserved relative to those of serine/threonine residues (XXRXRXXS/TXX . We observed the phosphorylation of hTERT peptide by the human melanoma cell lysate or the activated recombinant Akt kinase proteins in vitro. With the treatment of the growth factor deprivation or okadaic acid, we also observed the up-regulation of both hTERT peptide phosphorylation and the telomerase activity. We noticed that Wortmannin down-regulates hTERT peptide phosphorylation and telomerase activity together. In addition, we observed the enhancement of telomerase activity with the pretreatment of Akt kinase in vitro. Thus, these observations suggest that Akt kinase enhances human telomerase activity through phosphorylation of hTERT subunit as one of its substrate proteins.