The diagnosis of metastatic clear cell renal cell carcinoma (CC-RCC) can be difficult because of its morphologic heterogeneity and the increasing use of small image-guided biopsies that yield scant diagnostic material. This is further complicated by the degree of morphologic and immunophenotypic overlap with nonrenal neoplasms and tissues, such as adrenal cortex. In this study, a detailed immunoprofile of 63 adrenal cortical lesions, which included 54 cortical neoplasms, was compared with 185 metastatic CC-RCCs using traditional [anticalretinin, CD10, antichromogranin, antiepithelial membrane antigen, anti-inhibin, antimelanA, anticytokeratins (AE1/AE3 and AE1/CAM5.2), antirenal cell carcinoma marker, and antisynaptophysin)] and novel [anticarbonic anhydrase-IX, antihepatocyte nuclear factor-1b, antihuman kidney injury molecule-1 (hKIM-1), anti-PAX-2, anti-PAX-8, antisteroidogenic factor-1 (SF-1), and anti-T-cell immunoglobulin mucin-1] antibodies. Tissue microarray methodology was used to simulate small image-guided biopsies. Staining extent and intensity were scored semiquantitatively for each antibody. In comparing different intensity thresholds required for a “positive” result, a value of ≥2+ was identified as optimal for diagnostic sensitivity/specificity. For the distinction of adrenal cortical lesions from metastatic CC-RCCs, immunoreactivity for the adrenal cortical antigens SF-1 (86% adrenal; 0% CC-RCC), calretinin (89% adrenal; 10% CC-RCC), inhibin (86% adrenal; 9% CC-RCC), and melanA (86% adrenal; 10% CC-RCC) and for the renal epithelial antigens hKIM-1 (0% adrenal; 83% CC-RCC), PAX-8 (0% adrenal; 83% CC-RCC), hepatocyte nuclear factor-1b (0% adrenal; 76% CC-RCC), epithelial membrane antigen (0% adrenal; 78% CC-RCC), and carbonic anhydrase-IX (3% adrenal; 87% CC-RCC) had the most potential use. Use of novel renal epithelial markers hKIM-1 (clone AKG7) and/or PAX-8 and the adrenocortical marker SF-1 in an immunohistochemical panel for distinguishing adrenal cortical lesions from metastatic CC-RCC offers improved diagnostic sensitivity and specificity.