The prothymosin alpha kinase (ProTalphaK) is an apparently novel enzyme that is responsible for the phosphorylation of prothymosin alpha (ProTalpha), involved in the proliferation of mammalian cells. The present study investigated the properties of this enzyme. ProTalphaK is more effectively activated by Mn2+ than by other divalent cations, and its activity is unaffected by RNA. Its principal substrate in proliferating cells appears to be ProTalpha. Both in vivo and in vitro, it is unable to phosphorylate the peptides thymosin alphaI and thymosin alphaII, derived from the amino terminus of ProTalpha, despite the fact that the sites of phosphorylation of ProTalpha are contained within this part of its sequence. In trials in vivo, inhibition of gene expression abolished both phosphorylation of ProTalpha and ProTalphaK activity. ProTalphaK is located in the cytosolic fractions throughout the cell cycle. Its activity, which is dependent on cell proliferation, increases markedly during S phase and begins to decline as the cell enters G2. Studies of the effects of activators and inhibitors of protein kinases involved in signal transduction pathways suggest that ProTalphaK is activated by phosphorylation in a mitogen-initiated pathway that is dependent on PKC; however, PKC does not itself phosphorylate ProTalphaK, which is therefore presumably phosphorylated by another kinase.