A human cDNA was cloned that encodes a DNA-binding protein (SATBl) that is expressed predominantly in thymus and binds selectively to the nuclear matrix/scaffold-associating DNAs (MARslSARs). Missing nucleoside experiments showed that SATBl selectively binds in a special AT-rich sequence context where one strand consists of mixed A’s, T’s, and C’s, excluding G’s (ATC sequences). When this feature is destroyed by mutation, SATBl binding is greatly reduced even if the direct contact sequence remains intact. Conjunctional SATBl-binding sequences become stably unpaired in supercoiled DNA. Specific mutations that diminish the unwinding potential greatly reduce SATBl binding. However, SATBl does not bind single-stranded DNA. Chemical interference assays show that SATBl binds along the minor groove with very little contact with the bases. This suggests that SATBl recognizes the ATC sequence indirectly through the altered sugar-phosphate backbone structure present in the double-stranded DNA.