Nuclear matrix was isolated from cultured human fibroblasts by extraction of nuclei with 2 M NaCl. Electron microscopic observation on the isolated nuclear matrix revealed a fine network structure. The matrix fraction contained approximately 15% of total nuclear DNA and the matrix DNA was about 3- to 4-fold enriched in transcriptionally active collagen I (alpha 2) gene sequences, whereas transcriptionally inactive beta-globin gene sequences were not enriched. The nuclear matrix contained two major proteins of 65,000 and 45,000 daltons (pI 5.9 and 5.6, respectively). The DNA-binding activity of these nuclear matrix proteins was examined by Western blotting or by nitrocellulose filter-binding assay using cloned specific gene probes. The results suggest that there is no base sequence specificity in the binding, and that protein species of 60,000 to 200,000 daltons showed DNA-binding activity. These results indicate that association of transcribing genes with the nuclear matrix may reflect the functional state of the genes and may not be determined solely by the base sequence specificity of DNA binding. The nuclear matrix protein of 65,000 daltons was phosphorylated in vivo, and was the main substrate for protein kinase(s) associated with the nuclear matrix.