The transcription factor HIF-1α has been identified as a key regulator in the cellular and systemic response to hypoxia. Because hypoxia is frequently associated with acidosis, like in ischemia or tumour growth, we studied the impact of acidosis on the expression of the HIF-1α and HIF-2α proteins and that of the three HIF target genes carbonic anhydrase-9 (CA-9), glucose transporter-1 (Glut-1) and erythropoietin (EPO). Since the HIF-prolyl hydroxylases (PHD) modulate cellular HIF-α protein levels we also investigated changes in PHD mRNA expression under hypoxia and acidosis. HIF-1α immunoblots revealed, depending on the cell line investigated, a moderate induction of HIF-α protein levels by acidosis in normoxia (Hep3B cells) or hypoxia (HeLa cells). Concordantly, the activity of HIF-driven luciferase reporters was slightly enhanced at pH 7.0. In contrast, HIF target genes exhibited divergent responses to acidosis: basal and hypoxia-induced CA-9 mRNA levels were further increased, whereas hypoxic EPO mRNA induction was attenuated, and Glut-1 mRNA levels were not altered by acidosis. Except from a small increase of hypoxia-induced PHD3 mRNA levels in HeLa cells, there was also no significant effect of acidosis on PHD expression. In conclusion, albeit HIF protein levels slightly induced by acidosis and the inconsistent regulation of HIF target genes under acidosis suggest additional, yet unidentified pH-sensitive factors to be involved in the regulation of these genes.