In the present study, we investigated the role of the extracellular signal-regulated protein kinase (ERK) in regulation of the hypoxia marker, carbonic anhydrase IX (CAIX). U0126, a specific inhibitor of MEK1/2, downregulated CAIX expression induced by true hypoxia and cell density. CA9 promoter activity was similarly affected. Mapping of the U0126 effect revealed that both critical elements within the CA9 promoter, the hypoxia response element and the juxtaposed SP1-binding PR1, were inhibited. This confirmed that ERK signaling modulates CA9 promoter activity via its effects on hypoxia inducible factor-1 (HIF-1) and SP1. Further analysis of the U0126 effect on HIF-1-dependent transcription in MCF-7 cells identified p300, a transcriptional co-activator of HIF-1, as the target of ERK. Constitutively increased ERK activity in isogenic fibrosarcoma cell lines did not cause increased cell density-dependent CAIX expression/CA9 promoter activity. In HeLa cells, an inverse correlation between cell density-induced CAIX expression and ERK activation was observed: sparse cultures did not express CAIX and displayed high ERK activation, whereas CAIX expression in dense cultures was associated with low ERK activation. Collectively, our data do not support any quantitative relationship between ERK activation and CAIX expression. Thus, although ERK signaling is required for optimal CAIX expression, our data are consistent with a model in which constitutive basal ERK activity plays an auxiliary role in CA9 promoter transactivation by modulating activity of the transcription factor SP1 and the transcriptional co-activator p300. © 2005 Wiley-Liss, Inc.