To evaluate the significance of immunocytochemistry (ICC) performed on Thin-Layer Cytology (TLC) preparations. During the period August 2004 to May 2010, 5,736 consecutive fine needle aspiration biopsies were processed by both conventional smears and TLC preparations, and 180 cases required further immunocytochemical staining to resolve diagnostic questions. TLC preparations were processed with the Thin-Layer Cytology 2000TM Method (Cytyc Corp, Marlborough, MA). Conventional smears and TLC preparations were stained with HE. ICC staining was carried out on TLC slides. All 180 cases were processed successfully for ICC. In 161 cases (89.5%), ICC staining resulted in a definitive diagnosis, while in 19 cases (10.5%) the diagnosis was still inconclusive. About 148 (82.2%) of the cases also had tissue available for histological examination. Accepting the histologic findings as the true diagnosis, the sensitivity and specificity of the ICC diagnoses were 98.6 and 97.3%, respectively. ICC can be successfully applied on TLC preparations, and its results can be very useful for making diagnoses on fine needle aspiration (FNA) biopsies. Diagn. Cytopathol. 2012. © 2011 Wiley Periodicals, Inc.