We investigated the expression of H1, H2 relaxin and INSL-3, mRNA and protein, and LGR7 and LGR8 transcripts in human C-cell hyperplasia, primary medullary thyroid carcinoma (MTC) tissues, MTC metastases, and the human MTC-TT and mouse MTC-M cell lines. Relaxin-like peptide hormones were detected in C-cell hyperplasia and in MTC tissues, but were absent in human normal parafollicular C-cells of benign goiter tissues. In contrast to calcitonin, mRNA, and immunoreactive protein, no differences in the expression of relaxin and INSL3 were observed in MTC tissues of different pTNM classification or between primary and metastatic MTC tissues studied. All MTC tissues constitutively expressed LGR7 and LGR8 transcripts. Thus, relaxin-like hormones appear to be present early during C-cell hyperplasia and potentially functional relaxin/INSL3 ligand-receptor systems are present in human MTC tissues and in MTC cell lines.