The chemically inert trivalent ion cobalt(III) hexaammine, Co 3+(NH 3) 6, was found to exert polyamine-like effects in enhancing certain cyclic nucleotide-independent protein kinase reactions catalyzed by nuclear enzyme preparations from rat ventral prostate or liver. At 1 mM, Co 3+(NH 3) 6 stimulated chromatin- and also non-histone-protein-associated kinase activities with partially dephosphorylated phosvitin as substrate by 38% and 72% respectively, whereas chromatin-associated kinase-catalyzed phosphorylation of lysine-rich histones was not affected under the same conditions. 32P incorporation (from γ- 32P-ATP) into endogenous protein substrates of chromatin or non-histone protein fractions catalyzed by their erdogencus kinase activity was increased by 47% and 153%, respectively. These effects of Co 3+(NH 3) 6 were similar to those produced by 1mM spermine. Autoradiographic analysis of endogenous 32P-labelled nonhistone proteins revealed similar enhancements of the phosphorylation of several of the same proteins, induced by 1mM spermine or 1 mM Co 3+(NH 3) 6 or 2mM spermidine. The stimulatory actions of polyamines or Co 3+(NH 3) 6 were not mimicked by raising the ionic strength by addition of comparable concentrations of NaCl. The effects of 1 mM spermine and of 1 mM Co 3+(NH 3) 6 tested separately were not additive. Phosphorylation of lysine-rich histones by beef heart cyclic AMP-dependent protein kinase was not affected by polyamines or Co 3+(NH 3) 6 Various findings hint that the enhancement of cyclic nucleotide-independent kinase-catalyzed phosphorylation of certain protein substrates by spermidine, spermine and Co 3+(NH 3) 6 is primarily due to interaction of these cations with appropriate protein substrates affecting their conformational status. Further, these effects of polyamines may be a reflection of their cationic charge properties rather than being dependent on any particular conformations assumed by the polyamines.