The evolutionally conserved MAPK Sty1 and bZIP transcriptional activator Atf1 are known to play a pivotal role in response to the reactive oxygen species in S. pombe. However, it is unclear whether all of the H(2)O(2)-induced genes are directly regulated by the Sty1-Atf1 pathway and involved in growth fitness under H(2)O(2)-induced stress conditions. Here we present the study on ChIP-chip mapping of the genomic binding sites for Sty1, Atf1, and the Atf1's binding partner Pcr1; the genome-wide transcriptional profiling of the atf1 and pcr1 strains in response to H(2)O(2); and the phenotypic assessment of approximately 90 Atf1/Pcr1-bound or unbound genes for growth fitness under H(2)O(2) conditions. ChIP-chip analysis shows that Atf1 and Pcr1 binding sites are overlapped in the genome and constitutively present before H(2)O(2) stress. On the other hand, Sty1 recruitment primarily occurs at the Atf1/Pcr1 binding sites and is induced by H(2)O(2). We found that Atf1/Pcr1 is clearly responsible for the high-level transcriptional response to H(2)O(2). Furthermore, phenotypic assessment indicates that among the H(2)O(2)-induced genes, Atf1/Pcr1-bound genes exhibit a higher likelihood of functional requirement for growth fitness under the stress condition than the Atf1/Pcr1-unbound genes do. Notably, we found that the Atf1/Pcr1-bound genes regardless of their responsiveness to H(2)O(2) show a high probability of requirement for growth fitness. Together, our analyses on global mapping of protein binding sites, genome-wide transcriptional profiling, and phenotypic assessment provide insight into mechanisms for global transcriptional regulation by the Sty1-Atf1 pathway in response to H(2)O(2)-induced reactive oxygen species.