There has been much controversy about the presence of TNF-alpha within thyroid tissue. We therefore conducted a study to determine if TNF-alpha mRNA is present in thyroid tissue and thyroid-derived cells. Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was employed with a heterologous competitor fragment. Significantly lower levels of TNF-alpha mRNA were found in the autonomous nodules from patients with thyroid autonomy (TA; n = 4; 5.7 +/- 1.3 arbitrary units (AU) (mean +/- s.e.m.); P < 0.03) and in normal thyroid tissue (n = 2, 7.0 +/- 3.1 AU) compared with tissue from patients with Graves' disease (GD; n = 13; 27.9 +/- 10.3 AU), non-toxic multinodular goitre (NTG; n = 5; 20.9 +/- 5.8 AU) and perinodular tissue from TA patients (20.3 +/- 4.0 AU). Higher levels were detected in tissues from patients with Hashimoto's thyroiditis (HT; n = 2; 51.3 +/- 10.3 AU). Cultures of pure thyroid-derived fibroblasts (46 +/- 18 AU thyrocytes (33 +/- 8 AU), and the anaplastic thyroid carcinoma cell lines 8505 C (39 +/- 11 AU), SW 1736 (214 +/- 16 AU) and C643 (3 +/- 1 AU) showed significantly lower TNF-alpha mRNA levels than thyroid-derived lymphocytes (1650 +/- 32 AU). TNF-alpha was detected in the supernatants of unstimulated lymphocytes (22.1 +/- 1.1 pg/ml) and SW 1736 cells (3.5 +/- 0.9 pg/ml), but not in unstimulated fibroblasts and thyrocytes. Using an intracellular labelling technique in flow cytometry, the immunophenotype of stimulated TNF-alpha-positive lymphocytes was determined as predominantly CD3+CD45RO+. Our results suggest that TNF-alpha is present in the thyroid tissue of different thyroid disorders. Thyroid-derived lymphocytes are potential TNF-alpha producers and may thus locally influence thyroid function.