Fractionation of a 0.2 M NaCl nuclear extract from rat liver cells by both tubulin and DNA affinity chromatography steps allowed us to find three polypeptides interacting in vitro with both DNA and tubulin. A 22 kDa polypeptide was identified as a proteolytic fragment of High Mobility Group proteins 1 or 2 (HMG 1 or 2). Purified rat liver HMG 1 immobilized on nitrocellulose was found to bind radioiodinated dimeric tubulin through its central B domain. The C domain of HMG 1 appeared to play a negative role in this association process. Soluble HMG 1 depleted of its C-terminal domain interacted with tubulin immobilized on an agarose gel and with microtubules formed from purified tubulin. In contrast, undigested HMG 1 did not interact with tubulin in these conditions. The modification of HMG 1 with amine by 1-ethyl-3-(dimethylaminopropyl)carbodiimide which caused the neutralization of the C domain carboxyl groups restored the ability of HMG 1 to interact with microtubules. These results show that: (a) HMG 1, through its central B domain, binds to both assembled and non-assembled tubulin in vitro and (b) the C-terminal domain of HMG 1 exerts a negative regulatory action on the interaction.