Mammalian high mobility group HMG-I/Y chromatin proteins bind to the minor groove of A.T-rich DNA sequences with high affinity both in vivo and in vitro. Topoisomerase I-mediated relaxation assays, analyzed by one- and two-dimensional agarose gel electrophoresis, indicate that binding of recombinant human HMG-I/Y to closed circular DNA introduces positive supercoils at low protein to nucleotide molar ratios and negative supercoils at higher ratios. This is interpreted to mean that HMG-I/Y binding initially causes bending of the DNA helix followed by unwinding of the helix. In contrast, binding of another minor groove binding ligand, netropsin, introduces positive supercoils only. An in vitro produced mutant HMG-I/Y protein lacking the negatively charged carboxyl-terminal domain binds A.T-rich DNA approximately 1.4-fold better than the native protein, yet it is estimated to be 8-10-fold more effective at introducing negative supercoils. This finding suggests that the highly acidic C-terminal region of the HMG-I/Y protein may function as a regulatory domain influencing the amount of topological change induced in DNA substrates by binding of the protein. Footprinting of HMG-I/Y on negatively supercoiled A.T-rich DNA using diethylpyrocarbonate suggests that the protein is able to recognize, bind to, and alter the conformation of non-B-form DNA.