Three protein binding sites have been identified in the upstream region of the sericin-1 gene. Two of them, SA and SC sites, have been known as putative cis-acting elements. Using synthetic oligonucleotides of these binding sites, it was found that silk gland factor-1 (SGF-1) binds to the SA site, and silk gland factor-3 (SGF-3) binds to the SC site but not to a mutated SC site, SCM. Tissue distribution of the two factors was different. SGF-3 is present abundantly in the middle silk gland (MSG) where the sericin-1 gene is transcribed specifically but is also present in other cell types, though in a much less concentration. SGF-1 is observed very abundantly in the two parts of silk gland, MSG and posterior silk gland (PSG), but not in other cells. Templates containing multimerized SA or SC sites at -39 of the sericin-1 gene promoter were tested in MSG nuclear extracts. The SC multimer strongly activated transcription, while the mutant SCM multimer did not. The SA multimer also gave a slight stimulation of transcription. These results suggest that SGF-3 stimulates transcription through an interaction with the SC site, and SGF-1 does so weakly through the SA site.