The Hypoxia inducible factor-1 (HIF-1) is known as an important transcription factor in the context of cellular response to hypoxia. Although the structure of HIF-1a and its mechanisms are well known, numerous aspects concerning its regulation are still unexplored. One assumed reaction is the RNA-Interference of antisense variants of HIF-1a (aHIF-1a), which may enforce degradation in the nucleus or alternative splicing of HIF-1a. Therefore we aimed to explore expression and localization of two so far unknown aHIF-1a-variants under defined conditions in vitro and in vivo to enlarge the state of knowledge about the posttranscriptional regulation of HIF-1a. In in vitro experiments we succeeded to detect two novel aHIF-1a variants, terminating in exon 2 (aHIF-1aEx2) and exon 14 (aHIF-1aEx14) in different cell lines. We verified them as spliced antisense variants of the HIF1a-gene locus through sequencing and in silico analysis. The comparison of GAPDH and 7sk as housekeeping genes allowed us to identify aHIF-1aEx2 as nuclear and aHIF-1aEx14 as cytoplasmatic variant. In vitro trials revealed that both variants are differentially expressed after induced hypoxia (CoCl₂) and that aHIF-1aEx14 correlates significantly with HIF-1a mRNA with and without exon 14 (0,953 and 0,949). Exercise induced hypoxia in vivo (half marathon) showed no effect on expression manner of the two antisense RNAs. Due to specific sub-cellular localization, ubiquitary and differential expression we suggest here that these aHIF-1a-variants are presumably associated with the regulation of HIF1a.