Prostate-specific antigen (PSA) and human glandular kallikrein 2 (hK2) are two closely related kallikreins, primarily produced by the prostate. These serine proteases are now used as biomarkers for the diagnosis and management of prostate cancer. Until recently, PSA and hK2 were thought to be strictly expressed in the prostate; however, numerous studies confirmed their presence in various biological fluids as well as in many normal and malignant tissues. Using reverse transcription-polymerase chain reaction (RT-PCR), we screened RNA extracted from 26 different normal tissues and found that both genes are expressed in the thyroid. Subsequently, we analyzed 15 RNAs extracted from thyroid tissues (10 benign and 5 malignant lesions) and found that both kallikreins were expressed in five specimens (four benign lesions and one malignant). Sequencing of the PCR products confirmed the specificity of our experiments. Immunohistochemistry localized PSA in oxyphilic cells of thyroid tissue. These data confirm expression of both PSA and hK2 in thyroid tissue and suggest that oxyphils are the source of their production. The function of these two proteases in thyroid tissue is unknown.