The formation of parathyroid hormone (PTH) in the parathyroid gland occurs via two successive proteolytic cleavages from larger biosynthetic precursors. The initial product coded for by PTH mRNA is pre-proparathyroid hormone (PreProPTH), a polypeptide of 115 amino acids. Within 1 min of synthesis, the polypeptide, proparathyroid hormone (ProPTH), is formed as a result of the proteolytic removal of the NH2-terminal 25 amino acids from Pre-ProPTH. After a delay of 15-20 min, the NH2-terminal six-amino acid sequence of ProPTH is removed to give PTH of 84 amino acids. To investigate the subcellular sites in the parathyroid cell where the biosynthetic precursors undergo specific proteolytic cleavages, we examined, by electron microscopy autoradiography, the spatiotemporal migration of autoradiographic grains and, by electrophoresis, the kinetics of the disappearance of labeled Pre-ProPTH and the conversion of labeled ProPTH to PTH in bovine parathyroid gland slices incubated with [3H]leucine for 5 min (pulse incubation) followed by incubations with unlabeled leucine for periods up to 85 min (chase incubations). By 5 min, 85% of the autoradiographic grains were confined to the rough endoplasmic reticulum (RER). Autoradiographic grains increased rapidly in number in the Golgi region after 15 min of incubation; from 15 to 30 min they migrated within secretory vesicles still in the Golgi region and then migrated to mature secretory granules outside the Golgi area. Electrophoretic analyses showed that Pre-ProPTH disappeared rapidly (by 5 min) and that conversion of ProPTH to PTH was first detectable at 15 min and was completed by 30 min. At later times of incubation (30-90 min), autoradiographic grains within the secretion glanules migrated to the periphery of the cell and to the plasma membrane, in correlation with the release of PTH first detected by 30 min. We conclude that proteolytic conversion of Pre-ProPTH to ProPTH takes place in the RER and that subsequent conversion of ProPTH to PTH occurs in the Golgi complex.