Recent studies have shown that hypoxia-inducible factor1alpha (HIF1alpha) is ubiquitinated by an E3-ligase complex containing von Hippel-Lindau gene product (pVHL) after which it is targeted for proteasomal degradation. In this study, we showed that HIF1alpha was stabilized in the pVHL-deficient cell line 786-0 treated with a proteasome inhibitor or Co2+. This suggests that HIF1alpha is also ubiquitinated by a pVHL-independent pathway and that its stability is regulated by Co2+. Indeed, using the COS cell expression system, we confirmed that HIF1alpha is ubiquitinated at the N-terminal region by a pVHL-independent pathway and that its degradation is inhibited by Co2+. We also demonstrated that Co2+ binds to both PAS domains in the N-terminal region of HIF1alpha. These observations imply that the stability of HlF1alpha is regulated by an additional pathway through the cobalt binding of PAS domains. (C) 2003 Elsevier Science (USA). All rights reserved.