The Saccharomyces cerevisiae hemoglobin is a flavoprotein of unknown function. It shares extensive sequence homology with the globin of Candida as well as those of several bacterial species. We have studied its gene regulation in order to better understand its purpose in the cell. Transcriptional analyses indicate that, in sharp contrast to the bacterial globins of Vitreoscilla and Alcaligenes eutrophus, the S. cerevisiae globin message is induced during logarithmic growth and under oxygen-replete conditions. Transcription of the S. cerevisiae hemoglobin gene is positively regulated by the transcription factors heme-activated protein (HAP) 1 and HAP2/3/4, which respond to intracellular heme levels. Anaerobically, there is a low level, HAP-independent induction of hemoglobin mRNA. Unlike other systems influenced by the HAP2/3/4 transcription factor complex, no activation of hemoglobin expression by growth in non-fermentable carbon sources is observed. Flavohemoglobin gene disruption does not alter cell viability or growth in a variety of oxygen conditions and carbon sources. Physical and genetic mapping of the S. cerevisiae flavohemoglobin gene places it on chromosome seven near the formyltetrahydrofolate synthase (ADE3) locus. These data indicate that, despite the high degree of homology, the S. cerevisiae globin may have a function distinct from those proposed for bacterial globins.