The high mobility group (HMG) proteins of the AT-hook family (HMGA) lie downstream in regulatory networks with protein kinase C, Cdc2 kinase, MAP kinase, and casein kinase 2 (CK2) as final effectors. In the cells of the midge Chironomus, almost all of the HMGA protein (cHMGA) is phosphorylated by CK2 at two adjacent sites. 40% of the protein population is additionally modified by MAP kinase. Using spectroscopic and protein footprinting techniques, we analyzed how individual and consecutive steps of phosphorylation change the conformation of an HMGA protein and affect its contacts with poly(dA-dT).poly(dA-dT) and a fragment of the interferon-beta promoter. We demonstrate that phosphorylation of cHMGA by CK2 alters its conformation and modulates its DNA binding properties such that a subsequent phosphorylation by Cdc2 kinase changes the organization of the protein-DNA complex. In contrast, consecutive phosphorylation by MAP kinase, which results in a dramatic change in cHMGA conformation, has no direct effect on the complex. Because the phosphorylation of the HMGA proteins attenuates binding affinity and reduces the extent of contacts between the DNA and protein, it is likely that this process mirrors the dynamics and diversity of regulatory processes in chromatin.