Nuclear matrix is a nuclear protein-DNA superstructure believed to be the exclusive site of DNA replication, transcription, repair, and recombination. The attachment regions of chromatin loops to the nuclear matrix, called MARs, nest origins of replication, have transcriptional enhancer activity, and via their interaction with protein transcription factors may govern gene switch during development and tissue-specific gene expression. In this study the 967 bp MAR of the chicken alpha-globin gene is analyzed for the presence of hexanucleotides from a number (83 in total) of vertebrate protein transcription factors and core origins of replication. A total number of 760 hexanucleotides from factor sites or origins of replication were used for this search. We found that: (1) The occurrence of protein transcription factor binding sites overall on the MAR fragment as well as on the enhancer and promoter regions of other genes is only about 1.2-1.5 times higher than in random DNA, something consistent for all MAR and enhancer sequences examined. However, a high concentration (up to 2.7 times over random sequences) of hexanucleotide factor sites is observed on small stretches of the alpha-globin gene MAR. (2) Some regulatory protein binding sites are underrepresented whereas others are overrepresented, giving to an MAR a particular transcription factor flavor. (3) The DNA curvature map of the MAR sequence and the potential sites of positioned nucleosomes suggest the sites where a competition between core histone octamers and protein transcription factors for DNA might be found. This approach might provide a novel technique to diagnose for the regulatory or nonregulatory function of a stretch of DNA. Furthermore, MARs are proposed to constitute important regulatory elements of genes in addition to enhancers, promoters, silencers, locus control regions, and origins of replication. Additional parameters such as interaction of a transcription factor with other transcription factors fixed at vicinal sites, DNA methylation, intrinsic DNA curvature torsional strain, and nucleosome positioning might also determine the high-affinity binding of a transcription factor to its functional sites and its exclusion from or low affinity binding to other nonregulatory regions.