Parathyroid hormone (PTH) undergoes rapid proteolysis in the liver, which results in the appearance of multiple COOH-terminal fragments in plasma. Using reverse-phase high-performance liquid chromatographic (HPLC) techniques, we have shown that biologically active bovine PTH (bPTH) internally labeled with [3H]tyrosine is, like 125I-labeled bPTH, rapidly metabolized by isolated rat Kupffer cells in vitro to multiple COOH-terminal fragments that are chemically identical with those previously found in plasma after metabolism in vivo. Quantitation of specific carboxyl fragments in crude mixtures is achieved rapidly by direct HPLC analysis and is as precise as that achieved by Edman degradation. In addition, several different carboxyl fragments with identical NH2 termini were resolved, revealing a complexity not apparent in previous studies employing direct Edman degradation of such mixtures. Parallel studies with [[35S]Met]bPTH show the generation, by the Kupffer cells in vitro, of several labeled NH2-terminal fragments which undergo rapid further degradation in vitro. Thus, hepatic metabolism of PTH by Kupffer cells proceeds by an initial endopeptidase cleavage within the hormonal sequence in a manner compatible with the generation of biologically active NH2 fragments.