High mobility group box 1 (HMGB1) protein is the most abundant chromatin-associated non-histone protein expressed in all nucleated eukaryotic cells. We examined the phosphorylation of mammalian HMGB1 by testing the ability of the cyclin-dependent kinase 5 (Cdk5) to use as substrates native protein, either unmodified or in vivo acetylated and recombinant HMGB1. It turned out that Cdk5 was active on the in vivo acetylated HMGB1 only. We studied the effect of the phosphorylation on the 'architectural' properties of the acetylated HMGB1. The treatment with Cdk5 of the acetylated HMGB1 inhibited its capacity to induce DNA end-joining but had no effect on its ability to recognize distorted DNA structures.