In intraspecific crosses between cultivated rice (Oryza sativa) subspecies indica and japonica, the hybrid male sterility gene S24 causes the selective abortion of male gametes carrying the japonica allele (S24-j) via an allelic interaction in the heterozygous hybrids. In this study, we first examined whether male sterility is due solely to the single locus S24. An analysis of near-isogenic lines (NIL-F 1 ) showed different phenotypes for S24 in different genetic backgrounds. The S24 heterozygote with the japonica genetic background showed male semisterility, but no sterility was found in heterozygotes with the indica background. This result indicates that S24 is regulated epistatically. A QTL analysis of a BC 2 F 1 population revealed a novel sterility locus that interacts with S24 and is found on rice chromosome 2. The locus was named Epistatic Factor for S24 (EFS). Further genetic analyses revealed that S24 causes male sterility when in combination with the homozygous japonica EFS allele (efs-j). The results suggest that efs-j is a recessive sporophytic allele, while the indica allele (EFS-i) can dominantly counteract the pollen sterility caused by S24 heterozygosity. In summary, our results demonstrate that an additional epistatic locus is an essential element in the hybrid sterility caused by allelic interaction at a single locus in rice. This finding provides a significant contribution to our understanding of the complex molecular mechanisms underlying hybrid sterility and microsporogenesis.H YBRIDS between genetically divergent species often show abnormal phenotypes that reduce fitness, such as sterility, weakness, and inviability. Such hybrid characteristics, collectively called hybrid incompatibility, are assumed to play important roles in speciation by acting as postzygotic reproductive barriers. The genetic mechanisms of reproductive barriers and their implications in evolution have been studied using a variety of plant species including Helianthus (Rieseberg et al. 1996), Mimulus (Sweigart et al. 2006), Iris (Taylor et al. 2009), and rice (Sano 1990;Koide et al. 2008). These efforts have demonstrated that diverse mechanisms of hybrid incompatibility exist and have shed some light on the roles of these mechanisms in plant evolution. However, aside from a few cases, the molecules and networks that control hybrid incompatibility remain largely unknown. It is widely accepted that interactions between genetic loci (called epistasis) contribute to reproductive isolation mechanisms. Such genetic interactions most likely reflect the existence of molecular networks that control reproductive isolation. For example, an immunity system was found to be involved in hybrid incompatibilities resulting from intraspecific crosses in Arabidopsis thaliana (Bomblies et al. 2007;Alcazar et al. 2010).Because of its wide genetic diversity and well-characterized genetic base, cultivated rice (Oryza sativa L. 2n = 24) is a useful model for the study of hybrid sterility in plants. A number of hybrid sterility genes/Q...