The issue of whether histone H1 possesses specificity of binding to certain nucleotide sequences in DNA is of fundamental importance to the suggested role of the linker histone in the regulation of gene transcription. The purpose of the present study was to reinvestigate the specificity of binding of histone H1 to the putative nuclear factor I (NFI) recognition sequence suggested by a previous report in the literature. The interaction of purified mouse liver histone H1 with a synthetic oligonucleotide representing the natural NFI binding site from the adenovirus 2 origin of replication cloned in pBR322 has been studied by filter binding and a solid-phase procedure performed on nitrocellulose filter-immobilized protein dots. No indication of specific interactions of the lysine-rich histone H1 with the NFI recognition sequence was obtained.