The intracellular compartments where posttranslational processing of proTRH takes place have not been identified. Using AtT20 cells transfected with a complementary DNA for preproTRH, we have used purified antibodies that recognize the intact precursor, intermediate and end products of processing to identify the subcellular compartments in which cleavage occur. Further, pulse-chase experiments followed by subcellular fractionation were undertaken to determine the order of processing of proTRH during its transport to the secretory granules. Cells were homogenized by nitrogen cavitation and subjected to a centrifugation of 1.065 mg/ml density gradient of Percoll to separate secretory granules (SG) from rough endoplasmic reticulum (RER)/Golgi apparatus. The purity of the SG and RER fractions was assessed by assays of marker enzymes for mitochondria, RER, Golgi, and cytoplasm. ProTRH derived cryptic peptides and TRH in each fraction were determined by RIA. Golgi and SG fractions were subjected to polyacrylamide gel electrophoresis followed by extraction and RIA. Using the anti-pCC10 antiserum which recognizes intact (26 kd) as well as partially processed prohormone, the RER/Golgi fraction contained 0.3 pmol intact ProTRH and 0.2 pmol each 15 and 6 kilodalton (kDa) fragments; the SG contained the 15 kDa moiety (0.2 pmol) along with a 6 kDa (0.4 pmol) material but not the 26 kDa ProTRH. The SG were also enriched by 0.21 pmol pYE27 (PreproTRH 25-50), 0.23 pmol pFT (PreproTRH 53-74), 0.31 pmol pEH24 (PreProTRH 86-106), and 0.5 pmol TRH. None of these were present in the RER/Golgi. Pulse-chase studies also showed that the intact proTRH (26 kDa) precursor was only present in the RER/Gg fraction along with two of its N-terminal intermediate processing products, a 15 k mol wt peptide and a 6 k mol wt peptide, and two of its C-terminal processing products, a 16.5 k mol wt and a 9.6 k mol wt peptides. In addition, fully processed peptides as well as TRH were only detected in the neurosecretory granules. These observations suggest that after the initial conversion of proTRH in the RER/Golgi fraction, the peptides are delivered to the granules where processing to TRH and cryptic peptides takes place. Supporting this, our pulse-chase studies unequivocally showed that, pEH24, an end product of proTRH processing, was only produced in secretory granules. Thus, initial cleavage of the TRH precursor may be required for packing and sorting of the end products to occur.