Several splice variants exist of the 3a-subtype of human hypoxia-inducible factor (HIF-3a) whose expression and biological functions are largely unknown. In this study, eight transcripts of HIF-3a were cloned from OVCAR-3 mRNA which displayed distinct variability in three truncated versions of the first exon, a variable inclusion of the 3rd exon, and truncations at exons 8 or 10. Transfection studies revealed the expression of four variant-specific proteins which comprised one extended (V1b) and two truncated forms (V4, V8) of the established full-length isoform (V1a). Transcripts of these variants were detected in various human tissues with the V1 subtypes being the most abundant. In contrast, several tumor-derived cell lines showed highest mRNA levels of the V8 variant. HIF-2a responsive elements could be identified in the HIF-3a promoter which contributed to the upregulation of the V1 mRNA in OVCAR-3 during hypoxia. Transient expression of HIF-3a isoforms suppressed the activation by cotransfected HIF-1a or hypoxia of a HIF-responsive promoter construct, with the subtype V1b demonstrating the highest activity. Expression profiling after overexpression in OVCAR-3 cells also revealed a genuine transcriptional activity of HIF-3a variants on specific target genes. The subtypes V1a and V1b induced the expression of IGFBP1 which was also identified as a new target gene of HIF-1a and -2a. In contrast, the expression of HSPA1B was not influenced by classical HIFs, but was selectively induced by V1a, V1b and V8. Consequently, the major splice variants of HIF-3a emerge as transcriptionally regulated components of the HIF system with intrinsic transcriptional activities that specifically extend the spectrum of hypoxia-regulated genes.