In plants, chromosomal high mobility group (HMG) proteins have been identified in the HMGA family, containing A/T-hook DNA binding motifs, and in the HMGB family, containing an HMG-box DNA binding domain, that are considered architectural factors in chromatin. We have characterized the association of the HMGA protein, five different HMGB proteins, and the structure-specific recognition protein 1 (SSRP1) with maize chromatin by extraction experiments using NaCl, ethidium bromide, spermine, and distamycin A. The difference in the release of the proteins from chromatin by these reagents indicates that they are differentially associated with chromatin. This was confirmed by treatment of chromatin with micrococcal nuclease, demonstrating that the HMGA, HMGB2/3, and SSRP1 proteins are enriched in the highly nuclease-sensitive fraction of chromatin, which is likely to be transcriptionally competent. As examined by electrophoretic mobility shift analyses, the HMGA protein and the proteins containing an HMG domain (HMGB proteins and SSRP1) bind specifically to purified maize mononucleosomes that contain a histone octamer and approximately 165 bp of DNA. The mode of interaction with the nucleosomes differs for HMGA and HMGB proteins. In the case of the HMGB1 protein, the full-length protein is required for specific nucleosome binding, as the individual HMG-box DNA binding domain (which is sufficient for DNA interactions) interacts nonspecifically with the nucleosomes. Collectively, these findings indicate that HMGA, the various HMGB proteins, and SSPR1 are differentially associated with plant chromatin and may act as architectural factors in different nucleoprotein structures.