Aberrant promoter hypermethylation is a common mechanism for inactivation of tumor suppressor genes in cancer cells. To generate a global profile of genes silenced by hypermethylation in renal cell cancer (RCC), we did an expression microarray-based analysis of genes reactivated in the 786-0, ACHN, HRC51, and HRC59 RCC lines after treatment with the demethylating drug 5-aza-2 deoxycytidine and histone deacetylation inhibiting drug trichostatin A. Between 111 to 170 genes were found to have at least 3-fold up-regulation of expression after treatment in each cell line. To establish the specificity of the screen for identification of genes, epigenetically silenced in cancer cells, we validated a subset of 12 up-regulated genes. Three genes (IGFBP1, IGFBP3, and COL1A1) showed promoter methylation in tumor DNA but were unmethylated in normal cell DNA. One gene (GDF15) was methylated in normal cells but more densely methylated in tumor cells. One gene (PLAU) showed cancer cell-specific methylation that did not correlate well with expression status. The remaining seven genes had unmethylated promoters, although at least one of these genes (TGM2) may be regulated by RASSF1A, which was methylated in the RCC lines. Thus, we were able to show that up-regulation of at least 6 of the 12 genes examined was due to epigenetic reactivation. The IGFBP1, IGFBP3, and COL1A1 gene promoter regions were found to be frequently methylated in primary renal cell tumors, and further study will provide insight into the biology of the disease and facilitate translational studies in renal cancer.