Mammalian cells are able to sense oxygen and regulate a number of genes in response to hypoxia. The transcription factor Hypoxia Inducible Factor-1 (HIF-1) was identified as an important key component of the hypoxia signaling pathway. HIF-1 is a heterodimer composed of two members of the basic helix-loop-helix transcription factor superfamily containing a PAS (PER-ARNT-SIM) domain: HIF-1alpha and HIF-1beta/ARNT. During the cloning by reverse transcriptase-polymerase chain reaction of the human HIF-1alpha subunit, we isolated two cDNA clones which corresponded to alternative splicing of the HIF-1alpha gene. Polymerase chain reaction analysis and sequencing revealed that both clones possessed three additional base pairs between exons 1 and 2. Also, one of them lacked 127 base pairs corresponding to exon 14. We demonstrate that the mRNA of this truncated form is expressed in several human cells lines and human skin but apparently not in rodents. When transfected in HEK 293 cells, the corresponding 736 amino acid protein (HIF-1alpha(736)) is regulated by hypoxia in a similar manner as the full-length HIF-1alpha (HIF-1alpha(FL)). In luciferase transfection assays, both recombinant proteins HIF-1alpha(736) and HIF-1alpha(FL) dimerize with HIF-1beta/ARNT and activate the VEGF promoter upon hypoxia. However, the shorter HIF-1alpha isoform is 3-fold less active than HIF-1alpha(FL), a result consistent with the lack of the C-terminal transactivation domain. As expected, this small isoform can compete with the endogenous and transfected full-length HIF-1alpha. Altogether, these results suggest that the HIF-1alpha(736) isoform modulates gene expression upon hypoxia.