Spermine-binding protein (a rat ventral prostatic protein with high affinity for spermine) was phosphorylated in situ through the action of intrinsic cellular protein kinase(s), suggesting it to be a phosphoprotein in vivo. The purified protein served as a substrate in a number of cyclic AMP-independent protein kinase reactions in vitro, but not for cyclic AMP-dependent, Ca2+ + calmodulin-dependent or Ca2+ + phospholipid-dependent protein kinases. Available data indicate that at least one of the cyclic AMP-independent protein kinases (cytosolic protein kinase C2) may be physiologically relevant in mediating the phosphorylation of this protein. The phosphorylation reaction was stimulated several-fold in the presence of spermine. Spermidine was somewhat less effective, whereas putrescine, cadaverine and 1,6-hexanediamine were minimally active. Phospho amino acid analysis of 32P-labelled spermine-binding protein indicated that phosphoserine was the only labelled phospho amino acid. Spermine-binding protein did not undergo autophosphorylation, or modify the stimulative effect of spermine on the phosphorylation of other substrates such as non-histone proteins. In situ the phosphorylation of spermine-binding protein in tissue from castrated rats was markedly diminished as compared with the normal. Since the phosphorylation of spermine-binding protein appears to be mediated by cyclic AMP-independent protein kinase(s) whose activity in the prostate is under androgenic control, it is suggested that androgen-dependent modulation of the protein kinase(s) exerts a regulatory control (via phosphorylation-dephosphorylation) on the spermine-binding activity and stability of this protein in vivo. Further, since this protein is a substrate for only the cyclic AMP-independent protein kinases, it could serve as a tool for the investigation of such kinases.