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The stimulation of DNA synthesis in primary cell cultures of chicken chondrocytes by parathyroid hormone was studied by assaying [3H]thymidine incorporation into DNA. Optimal assay conditions were determined by varying cell age, plating density, and incubation time. Under these conditions DNA synthesis was significantly stimulated by parathyroid hormone (PTH) and some of its fragments: cells treated with human (h)PTH(1–84), bovine (b)PTH(1–34) and [Nle8,18,Tyr34]bPTH(3–34)amide and hPTH(13–34) displayed 2.6-fold enhanced [3H]thymidine incorporation in a dose-dependent manner. The fragment hPTH(28–48) led to a similar stimulation, whereas [Tyr43]hPTH(43–68) and [Tyr52,Asp76]hPTH(52–84) had no effect. Using a series of synthetic hPTH peptides covering the central region of the hormone molecule (residues 25–47), we could delimitate further this putative mitogenic functional domain to a core region between amino acid residues 30 and 34. The effect of PTH on [3H]thymidine incorporation could not be mimicked by forskolin, indicating that the corresponding signal is not mediated by cAMP. It is, however, inhibited by EGTA and cannot be provoked in the absence of calcium ions in the medium. Therefore, the results presented indicate a hitherto unidentified functional domain of PTH in the central part of the molecule which exerts its mitogenic effect on chondrocytes in a cAMP-independent manner but seems to involve calcium ions for signal transduction.