Adrenocortical carcinomas (ACC) are uncommon tumors of the adrenal cortex that are known to follow an aggressive clinical course. The distinction of these tumors from other neoplasms may sometimes prove difficult due to overlapping clinical, morphologic, and even immunophenotypical features. To this end, we performed a comprehensive immunohistochemical analysis using traditional and novel markers in 40 cases of ACC. An immunohistochemical panel consisting of 10 traditional and novel antibodies was applied to whole tissue sections of ACC including high-molecular weight cytokeratin (HMWCK), low-molecular weight cytokeratin (CAM5.2), inhibin-α, melan A, chromogranin A, synaptophysin, calretinin, steroid receptor coactivator-1 (SRC-1), Pax8, and Ki67. The percentage of positive tumor cells as well as the intensity of staining were evaluated and scored; for Ki67 the percentage of positive tumor cells was recorded. Positive staining was observed for SRC-1 (39/40; 97.5%), inhibin-α (37/40; 92.5%), calretinin (32/40; 80%), synaptophysin (29/40; 72.5%), melan A (26/40; 65%), and CAM5.2 (9/40; 22.5%). Rare cases showed positivity for chromogranin A (2/40; 5%) and Pax8 (1/40; 2.5%). None of the cases showed any reactivity with HMWCK. The Ki67 index ranged from <5% to 20%. We conclude that there is no single specific marker to reliably distinguish ACC from other primary or metastatic neoplasms. However, a combination of immunohistochemical stains in a panel consisting of SRC-1, inhibin-α, calretinin, and HMWCK may be of aid in the differential diagnosis of these tumors. In addition, Pax8 is only rarely positive in ACC, which is a useful tool in their separation from renal neoplasms.