Hypoxic induction of erythropoietin (Epo) and other oxygen-dependent genes is mediated by the hypoxia-inducible factor-1 (HIF-1), a heterodimeric transactivator consisting of an alpha and a beta subunit. We previously found that the mouse gene encoding HIF-1alpha harbors two alternative first exons (I.1 and I.2), giving rise to two different HIF-1alpha mRNA isoforms. Here, we show by RNase protection analysis that the exon I.1-derived mRNA isoform is differentially expressed in mouse tissues, being highest in kidney, tongue, stomach, and testis, but undetectable in liver, whereas the exon I.2 mRNA isoform is ubiquitously expressed. Sequence and methylation analysis showed that, in contrast to exon I.1, exon I.2 resides within a region showing typical features of a CpG island, known to be associated with the 5' end of housekeeping genes. We identified a 232-bp minimal exon I.2 promoter that strongly induced reporter gene expression in mouse L929 fibroblasts and Hepa1 hepatoma cells. In contrast to L929 cells, the exon I.1 promoter was inactive in Hepa1 cells and hypoxic exposure (1% O2) markedly reduced exon I.2 promoter activity in Hepa1 cells. Prolonged exposure of mice to hypoxia (7.5% O2 for up to 72 hours) also caused a decrease in liver HIF-1alpha mRNA, whereas aldolase mRNA levels increased. These findings might be related to the relatively low Epo levels in the adult liver.