The post-translational processing of prothyrotropin-releasing hormone (pro-TRH25-255) has been extensively studied in our laboratory, and the processing pathway to mature TRH has been elucidated. We have also demonstrated that recombinant PC1 and PC2 process partially purified pro-TRH to cryptic peptides in vitro and that pro-TRH and PC1 mRNAs are coexpressed in primary cultures of hypothalamic neurons. To further define the role of each convertase, and particularly PC1 and PC2, in pro-TRH processing, recombinant vaccinia viruses were used to coexpress the prohormone convertases PC1, PC2, PACE4, PC5-B, furin, or control dynorphin together with rat prepro-TRH in constitutively secreting LoVo cells or in the regulated endocrine GH4C1 cell line. Radioimmunoassays from LoVo-derived secreted products indicated that furin cleaves the precursor to generate both N- and C-terminal intermediates. PC1, PC2, and PACE4 only produced N-terminal intermediates, but less efficiently than furin. In GH4C1 cells, PC1, PC2, furin, PC5-B, and PACE4 produced both N-terminal and C-terminal forms. Significantly, TRH-Gly and TRH were mostly produced by PC1, PC2, and furin. Utilizing gel electrophoresis to further analyze the cleavage specificities of PC1 and PC2, we found that PC1 seems primarily responsible for cleavage to both intermediates and mature TRH, since it generated all products at significantly higher levels than PC2. The addition of 7B2 to the coinfection did not augment the ability of PC2 to cleave pro-TRH to either N- or C-terminal forms.