Based on evidence that the von Hippel-Lindau (VHL) tumor suppressor protein is associated with polysomes and interacts with translation regulatory factors, we set out to investigate the potential influence of pVHL on protein translation. To this end, renal cell carcinoma (RCC) cells that either lacked pVHL or expressed pVHL through stable transfection were used to prepare RNA from cytosolic (unbound) and polysome-bound fractions. Hybridization of cDNA arrays using RNA from each fraction revealed a subset of transcripts whose abundance in polysomes decreased when pVHL function was restored. The tumor necrosis factor alpha (TNF-alpha) mRNA was identified as one of the transcripts that preferentially associated with polysomes in pVHL-deficient cells. Additional evidence that the TNF-alpha mRNA was a target of translational repression by pVHL was obtained from reporter gene assays, which further revealed that pVHL's inhibitory influence on protein synthesis occurred through the TNF-alpha 3'-untranslated region. Our findings uncover a novel function for the pVHL tumor suppressor protein as regulator of protein translation.