Heme regulation of the activity of diverse proteins was thought to be mediated by heme-responsive motifs (HRMs). The yeast transcriptional activator Hap1 contains seven HRMs: HRM1-7. Three copies of a 17-amino-acid repeat are also located in the region encompassing HRM1 to -6. We examined the effects of these HRMs and repeats on heme regulation of Hap1 activity by deletion analysis and by Ala substitutions of key residues. We found that the effect of mutation or deletion of one HRM or 17-amino-acid repeat on Hap1 heme responsiveness is different from the effect of mutation or deletion of another HRM or repeat. Our data suggest that HRM7 plays a dominant role in mediating heme activation of Hap1 in heme-sufficient cells while HRM1-6 may scavenge heme and cause a low level of Hap1 activation in heme-deficient cells. These results may help in understanding the roles of HRMs in other hemoproteins.