Thyroid dysfunctions are common in chronic hepatitis C virus (HCV) infection. HCV-RNA has been detected by reverse-transcription polymerase chain reaction (PCR) in thyroid from HCV infected patients with acquired immunodeficiency syndrome. However, morphological evidence of HCV replication in thyroid cells from immune competent patients has not been provided. In situ hybridization and real-time-PCR were used to analyze HCV-RNA replication in thyroid tissue from 11 patients (3 anti-HCV, serum HCV-RNA positive; 8 anti-HCV negative). Genomic and antigenomic HCV-RNA was detected in the thyroid of the 3 anti-HCV positive patients at concentrations of 2.6 x 10(4), 1.7 x 10(4), and 8.6 x 10(3) copies/microg of total RNA (genomic) and 3.2 x 10(2), 4.3 x 10(3) and 2.9 x 10(2) HCV-RNA copies/microg of total RNA (antigenomic). No HCV-RNA was detected in the thyroid tissue of the 8 anti-HCV negative patients. Presence of genomic/antigenomic HCV-RNA in the 3 anti-HCV positive cases was confirmed by in situ hybridization. Signals were observed in the cytoplasm of the thyroid cells. In conclusion, the data obtained indicate that HCV may infect cells of the thyroid in immune competent patients with chronic HCV infection. The pathogenic implications of this finding merit further research.