We have recently identified two different human DNA-binding proteins, SMBP1 (35 kDa) and SMBP2 (17 kDa), that specifically interact with the direct repeats of the enhancer sequence in the squirrel monkey retrovirus long terminal repeat. Herein, we report several biochemical properties of the human DNA-binding proteins. SMBP1 and 2 recognized an overlapped sequence of the 5' region of the repeat which contains a palindrome of CCAATGG. Both proteins required divalent cations such as Mg2+ and Ca2+ for their specific DNA binding at the optimum concentration of 1 mM. SMBP2 is a thermostable protein that binds tightly to the DNA sequence even by treatment at 80 degrees C for 15 min. The SMBP2-DNA complex was also stable in the presence of 300 mM NaCl. The resistance of SMBP2 to heat and salt treatment is a prominent character distinguishable from SMBP1 and other known transcriptional factors. SMBP1 and 2 can be easily separated by heparin-agarose chromatography. These DNA-binding proteins were found to be present in nuclear extracts from several human cell lines including T cell, B cell, and epithelial cell.