High mobility group protein I (HMG-I) is a nonhistone chromosomal protein. The present study aims to examine phosphorylation of HMG-I by protein kinase C (PKC) and its effect on HMG-I's DNA binding activity. HMG-I, extracted and purified from rat brain was phosphorylated in vitro equally well by PKC alpha, beta, gamma and delta. Phosphoamino acid analysis indicated that both serine and threonine residues were phosphorylated. The nonphosphorylated HMG-I was shown to bind specifically to the fragment of DNA containing bp -708 to -458 of RC3 genomic DNA, which is abundant in A-T sequences. In contrast, phosphorylation of HMG-I by PKC resulted in an attenuation of binding to the DNA fragment. It is suggested that phosphorylation of HMG-I by PKC may regulate DNA binding activity of HMG-I, thereby possibly altering its biological functions.