Previous work from our laboratory described a protein that binds to single-stranded DNA in the early promoter of simian virus 40 in a sequence specific fashion. We have now used the gel retardation assay to search for similar sequence-specific single-strand-binding proteins for the promoter region of the rat albumin gene in nuclear extracts of rat hepatoma cells. Several proteins of this kind were detected, three of which are described in the present paper. Two of them bind specifically to the noncoding strand and the third one binds to the coding strand. The most abundant of these proteins binds to a pyrimidine stretch inside the coding region of the gene and appears to be homologous to the previously observed SV40-binding protein. Possible functions for sequence-specific single-strand-binding proteins in transcription are discussed.