To investigate the relationship between prohormone processing and sorting of mature polypeptides into nascent secretory vesicles, we recently developed a permeabilized cell system that supports both these reactions (Xu, H., and Shields, D. (1993) J. Cell Biol. 122, 1169-1184). Rat anterior pituitary GH3 cells expressing high levels of prosomatostatin (proSRIF) were incubated at 20 degrees C; this temperature prevented exit from the trans-Golgi network and inhibited proSRIF processing. Following the 20 degrees C block, the cells were mechanically permeabilized and incubated at 37 degrees C, and proSRIF processing was determined. Cleavage of proSRIF to the mature hormone required ATP hydrolysis and was inhibited by chloroquine, a weak base, or carbonyl cyanide m-chlorophenylhydrazone, a protonophore. This suggested that a proton gradient and/or an acidic pH facilitated by a vacuolar H(+)-ATPase was required for prohormone processing. We have now utilized the permeabilized cell system in conjunction with the antibiotic bafilomycin A1, a specific inhibitor of vacuolar H(+)-ATPases, to elucidate the role of acidic pH in prohormone processing. Here we report that (i) proSRIF processing was inhibited in vivo and in vitro by low concentrations of bafilomycin A1, confirming the involvement of a vacuolar type ATPase in prohormone processing; (ii) the ATP requirement for processing could be circumvented in vitro by incubating permeabilized cells at acidic pH in the presence of protonophores, indicating that an acidic pH rather than a H+ gradient is necessary for processing; and (iii) a pH of between 6 and 6.2 in the trans-Golgi network was optimal for proSRIF cleavage. We also demonstrate that prohormone convertase 2 exhibited temperature-dependent activity in which proSRIF processing was inhibited at 20 degrees C in vitro. This result explains our previous observation that prohormone processing is inhibited when intact cells are incubated at 20 degrees C.