Pre-proparathyroid hormone, discovered previously by the translation of messenger RNA from parathyroid glands in heterologous cell-free systems, is a polypeptide of 115 amino acids consisting of 25 amino acids added to the NH2 terminus of proparathyroid hormone (90 amino acids), the immediate biosynthetic precursor of parathyroid hormone (84 amino acids). We now have detected pre-proparathyroid hormone formation within intact parathyroid cells, thus providing direct evidence that it is a biosynthetic precursor of proparathyroid hormone. A radiolabeled protein synthesized by slices of bovine parathyroid glands during 1 to 10 min of incubation with [35S] methionine was identified that co-migrated on both urea-acetate and urea-SDS acrylamide gels with 3H-labeled pre-proparathyroid hormone prepared by mRNA translation in a cell-free system derived from wheat germ. The amount of radiolabeled protein reached a maximum 3 min after exposure of the tissue to[35S] methionine and decreased during a 3-min chase incubation with unlabeled methionine. It bound to antisera to both proparathyroid hormone and parathyroid hormone and contained 35S-labeled tryptic peptides that migrated identically with peptides of pre-proparathyroid hormone prepared from the wheat germ system. Radiolabeled proparathyroid hormone was identified in the tissue after 1 min of incubation with [35S] methionine. These findings indicate that pre-proparathyroid hormone is an early biosynthetic precursor of proparathyroid hormone in parathyroid tissue and that this precursor is converted to proparathyroid hormone within 1 min after completion of its synthesis on the rough endoplasmic reticulum.