The insulin-like hormone INSL-3, also named relaxin-like factor (RLF) or Leydig-derived insulin-like peptide (LEY-IL), is expressed in various reproductive tissues and is regarded a marker of differentiation in human testicular Leydig cells. Recently, we have identified differential expression of human INSL-3 in neoplastic Leydig cells and mammary epithelial cells suggesting an involvement of INSL-3 in tumor biology. Here we have investigated the expression of INSL-3 in human thyroid carcinoma cell lines and in the human thyroid gland which has been shown to express transcripts for the G protein coupled INSL-3 receptor LGR8. When we determined the expression of INSL-3 in eight human thyroid carcinoma cell lines, a novel INSL-3 splice variant containing a 95 bp out-of-frame insertion at the beginning of exon II of the INSL-3 gene was discovered. Treatment of the human anaplastic thyroid carcinoma cell line 8505C with diethylstilbestrol (DES) caused a significant dose-dependent transcriptional down-regulation of INSL-3 and a marked up-regulation of LGR8. Employing in situ hybridization to detect INSL-3 transcripts and specific rabbit antisera against the INSL-3 proteins, both INSL-3 isoforms were detected in patients with Graves' disease (n=10), follicular carcinomas (FTC; n=12), papillary carcinomas (PTC; n=9) and undifferentiated anaplastic carcinomas (UTC; n=15). By contrast, thyrocytes of all 15 benign goiter tissues studied were devoid of both INSL-3 isoforms, mRNA and protein. Our data indicate that INSL-3 hormone is up-regulated in hyperplastic and neoplastic human thyrocytes suggesting that the INSL-3 isoforms may serve as additional markers for hyperplastic and neoplastic human thyrocytes. In the anaplastic thyroid carcinoma cell line 8505C, the regulation of both INSL-3 and LGR8 by estrogen may be the first indication of a novel hormonally responsive, auto-/paracrine INSL-3 LGR8 ligand receptor system active in human thyroid carcinoma cells.