Regulation of vascular smooth muscle Ca2+channels by oxygen tension contributes importantly to hypoxic vasodilatation. We previously described the inhibitory effects of hypoxia on the recombinant human cardiac L-type Ca2+channel &agr;1Csubunit (hHT isoform) expressed in HEK 293 cells. We now demonstrate that hypoxia inhibits only one of the three naturally occurring splice variants of this channel that differ only in the C-terminal domain, permitting identification of a 71-amino acid insert in the C-terminal region of the channel that confers oxygen sensitivity. Selective restriction of the spliced insert allowed determination of a 39-amino acid region essential for oxygen sensing. This represents the first identification of the structural region of an ion channel required for sensing changes in oxygen tension.