We have encountered an increasing number of image-guided adrenal mass biopsies in which the differential diagnosis is adrenal cortical lesion versus pheochromocytoma. This distinction is sometimes difficult because of confounding clinical presentations, overlapping morphologies, and some degree of immunophenotypic overlap including focal staining with markers of purported lineage specificity. Interventional radiologists commonly use narrow gauge biopsy needles in this setting, which yield scant diagnostic tissue and further complicate pathologic evaluation. In this study, a detailed immunoprofile of 63 adrenal cortical lesions (3 adrenal rests, 6 adrenal cortical hyperplasias, 43 adrenal cortical adenomas, 4 adrenal cortical neoplasms of uncertain malignant potential, and 7 adrenal cortical carcinomas) was compared with 35 pheochromocytomas using traditional (calretinin, chromogranin, inhibin, melanA, and synaptophysin) and novel [steroidogenic factor-1 (SF-1), microtubule-associated protein 2, and mammalian achaete-scute homolog-1] antibodies, using tissue microarray technology to simulate small image-guided biopsies. Staining extent and intensity were each scored semiquantitatively for each antibody. A comparison of sensitivity and specificity using different intensity thresholds required for a “positive” result (≥1+ vs. ≥2+) was performed. Staining results based on a ≥1+ and (≥2+) intensity threshold were as follows: calretinin—95% (89%) in adrenal cortical lesions and 14% (0%) in pheochromocytomas; chromogranin—0% in adrenal cortical lesions and 100% in pheochromocytomas; inhibin—97% (86%) in adrenal cortical lesions and 6% (0%) in pheochromocytomas; microtubule-associated protein 2—29% (16%) in adrenal cortical lesions and 100% (89%) in pheochromocytomas; mammalian achaete-scute homolog-1—0% in both adrenal cortical lesions and pheochromocytomas; melanA—94% (86%) in adrenal cortical lesions and 6% (0%) in pheochromocytomas; SF-1—87% (86%) in adrenal cortical lesions and 0% in pheochromocytomas; synaptophysin—67% (59%) in adrenal cortical lesions and 100% in pheochromocytomas. Using an antibody panel consisting of chromogranin plus the nuclear antibody SF-1 and either calretinin or inhibin, while requiring a high-staining intensity threshold, helps to eliminate interpretative issues of artifactual or background reactivity, improves diagnostic sensitivity/specificity, and makes for an effective immunohistochemical approach in distinguishing adrenal cortical lesions from pheochromocytomas.