High mobility group protein HMG-I(Y) selectively binds to stretches of A.T-rich B-form DNA in vitro by recognition of substrate structure rather nucleotide sequence. Recognition of altered DNA structures has also been proposed to explain the preferential binding of this non-histone protein to four-way junction DNA as well as to restricted regions of DNA on random-sequence nucleosome core particles. Here we describe experiments that examine the influence of intrinsic DNA structure, and of structure imposed by folding of DNA around histone cores, on the binding of HMG-I(Y). As substrates for binding, we chose defined-sequence DNA molecules containing A.T-rich segments demonstrated previously to have very different structures in solution. These segments are either intrinsically bent (phase A.T tracts), flexible (oligo[d(A-T)]), or straight and rigid [oligo(dA).oligo(dT)]. DNase-I and hydroxyl radical footprinting techniques were employed to analyze protein binding to these DNAs either free in solution or when they were reconstituted into monomer or dinucleosomes in vitro. Results indicate that the DNA structure exerts a significant influence on HMG-I(Y) binding both when substrates are free in solution and when they are wrapped into nucleosomal structures. For example, when DNA is free in solution, HMG-I(Y) prefers to bind to the narrow minor groove of A.T sequences but sometimes also binds to certain GpC residues having narrowed major grooves that are embedded in such sequences. On the other hand, depending on the structure and/or orientation assumed by particular A.T-rich segments on the surface of reconstituted histone octamers, HMG-I(Y) binding site selection on individual nucleosomes differs considerably. Two observations are of particular importance: (i) HMG-I(Y) can preferentially bind to certain types of A.T-DNA located on the surface of nucleosomes; and (ii) HMG-I(Y) binding can induce localized alterations in the helical periodicity and/or rotational setting of DNA on the surface of some nucleosomes. The abilities of HMG-I(Y) suggests that in vivo the protein may play an important role in recognizing and altering the structure of localized regions of chromatin.