The induction by hypoxia of genes such as erythropoietin, vascular endothelial growth factor (VEGF), and glucose transporter-1 (Glut-1) is mediated in part by a transcriptional complex termed hypoxia-inducible factor-1 (HIF-1). Several lines of evidence have implicated protein phosphorylation in the mechanism of activation of HIF-1 by hypoxia. Recent reports have described the activation of the tyrosine kinase src by severe hypoxia, and a role in the induction of VEGF by severe hypoxia has been proposed. This led us to examine whether src and related kinases operated more widely in the hypoxic induction of HIF-1 and HIF-1-dependent genes regulated by hypoxia. Measurements of src kinase activity in cells exposed to varying severities of hypoxia showed activation by severe hypoxia (0.1% oxygen or catalyst induced anoxia), but not 1% oxygen. This contrasted with the marked induction of HIF-1 by exposure to 1% oxygen. Manipulations of src activity were produced by transient and stable transfection of Hep3B cells. Despite substantial changes in src activity, no alteration was seen in the normoxic or hypoxic expression of erythropoietin, VEGF, or Glut-1, or in the regulation of HIF-1-dependent reporter genes inducible by hypoxia. Similarly, we found that the expression of these genes in src- or c-src kinase-deficient cells did not differ from wild-type cells at either 1% oxygen or more severe hypoxia. These results indicate that src is not critical for the hypoxic induction of HIF-1, erythropoietin, VEGF, or Glut-1.