We have demonstrated that during opiate withdrawal, preprothyrotropin releasing hormone (preproTRH) mRNA is increased in neurons of the midbrain periaqueductal gray matter (PAG) while the concentration of TRH remained unaltered, suggesting that the processing of proTRH may be different in this region of the brain. The aim of the present study was to determine which of the proTRH‐derived peptides are affected by opiate withdrawal in the PAG. These changes were compared to other TRH‐containing areas such as the hypothalamic paraventricular nucleus (PVN), median eminence (ME) and the lateral hypothalamus (LH). Control and morphine‐treated rats 24 h following naltrexone‐precipitated withdrawal were decapitated and the brain microdissected. Pooled samples from each animal group were acid extracted, and peptides were electrophoretically separated then analyzed by specific radioimmunoassay. Opiate withdrawal caused a significant change in the level of some post‐translational processing products derived from the TRH precursor. In the PAG, opiate withdrawal resulted in an accumulation of the intervening preproTRH₈₃₋₁₀₆ peptide from the N‐terminal side of the prohormone, while the levels of the C‐terminal preproTRH_208‐‐285 peptide were reduced, with no change in preproTRH₂₅₋₅₀ or TRH, itself, as compared to control animals. Immunohistochemical analysis also showed significant increases in cellular preproTRH₈₃₋₁₀₆ peptide immunolabeling in the PAG. Opiate withdrawal in the lateral hypothalamus, unlike from the PAG, was accompanied by an increase in the concentration of TRH. In addition, western blot analysis showed that during opiate withdrawal, the mature form of the prohormone convertase 2 (PC2) increased only in PAG as compared with their respective controls. Thus, these results demonstrate a region‐specific regulation of TRH prohormone processing in the brain, which may engage PC2, further suggesting a role for specific proTRH‐derived peptides in the manifestations of opiate withdrawal.