The ROX1 gene encodes a product implicated in the regulation of heme-repressed and heme-induced genes in Saccharomyces cerevisiae. The gene has been cloned and shown to code for a 1.4-kilobase transcript. The cloned gene was used to construct a null mutant to determine the role of ROX1 in regulating the expression of several heme-regulated genes. Constitutive expression of ANB1 (a heme-repressed gene) was observed in the null strain, indicating that ROX1 codes for a repressor or a facilitator of repression. Enhancement of expression of CYC7 in the null strain indicated that the ROX1 factor is required for repression of CYC7 to its normal low level of expression, consistent with evidence that CYC7 has a hybrid heme-induced, heme-repressed regulatory mechanism. The null mutation had only a slight negative effect on expression of the heme-induced genes CYC1 and tr-1 (a heme-induced homolog of ANB1), suggesting that the ROX1 factor is not directly involved in their regulation despite the existence of an unusual rox1 mutation (rox1-a1) causing constitutive expression of this group. The respiratory competence of the null mutant indicates that ROX1 is not a respiratory factor. ROX1 expression was found to be induced by heme, indicating that the heme repression of ANB1 and its family is the result of a cascade in which heme induces a repression factor which keeps the family of heme-repressed genes inactive during aerobic growth. The rox1-a1 allele had earlier been shown to cause constitutive expression of the family of heme-induced respiratory genes. This allele was found to cause constitutive expression of the ROX1 transcript itself, indicating that ROX1 is in the major heme-induced regulon.