The 5'-flanking region of the rat insulin II gene (-448 to +50) is sufficient for tissue-specific expression. To further determine the tissue-specific cis-acting element(s), important sequences defined by linker-scanning mutagenesis were placed upstream of a heterologous promoter and transfected into insulin-producing and -nonproducing cells. Rat insulin promoter element 3 (RIPE3), which spans from -125 to -86, was shown to confer beta-cell-specific expression in either orientation. However, two subregions of RIPE3, RIPE3a and RIPE3b (defined by linker-scanning mutations), displayed only marginal activities. These results suggest that the two subregions cooperate to confer tissue specificity, presumably via their cognate binding factors.