The monoclonal antibody, HBME-1, generated against the microvillous surface of mesothelial cells, has been shown to have significant reactivity in histologic sections of follicular-derived thyroid malignancies. We examined the diagnostic utility of HBME-1 in thyroid fine-needle aspiration (FNA) specimens. Twenty-four aspirates from 23 patients were evaluated. Only cases with adequate cell blocks and tissue follow-up were studied. Immunocytochemical analysis was performed on air-dried, direct smears and on sections from Bouin's-fixed, paraffin-embedded cell blocks with a standard avidin-biotin peroxidase complex method with epitope retrieval. The same immunostaining technique was applied to the corresponding formalin-fixed tissue sections. Eight (57%) of the 14 malignant aspirates showed strong cytoplasmic and/or membrane immunoreactivity for HBME-1. The cell-block and direct-smear preparations were positive in five of seven papillary carcinomas (one follicular variant), one of one minimally invasive follicular carcinoma, and one of four hybrid tumors with mixed papillary and follicular features. An additional hybrid tumor case was focally positive only in the smear slide. The eighth positive case was an adenosquamous carcinoma of the larynx that invaded into the thyroid (smear preparation was negative for this case). The 10 benign lesions were negative. All of the malignant-tumor tissue sections were positive for HBME-1, and focal positivity was seen in 5 of 10 benign resection specimens. We conclude that a positive immunostain for HBME-1 on a thyroid FNA is supportive evidence that the lesion is a carcinoma, that a negative result for HBME-1 does not preclude the diagnosis of thyroid carcinoma, and that HBME-1 can be effectively applied to thyroid FNA specimens and can be a valuable adjunct in the cytologic diagnosis of thyroid malignancies.