--- - |2- Abstract The biosynthetic and endocytic pathways of secretory cells are characterized by progressive luminal acidification, a process which is crucial for posttranslational modifications and membrane trafficking. This progressive fall in luminal pH is mainly achieved by the vacuolar‐type‐H+ ATPase (V‐ATPase). V‐ATPases are large, evolutionarily ancient rotary proton pumps that consist of a peripheral V1 complex, which hydrolyzes ATP, and an integral membrane V0 complex, which transports protons from the cytosol into the lumen. Upon sensing the desired luminal pH, V‐ATPase activity is regulated by reversible dissociation of the complex into its V1 and V0 components. Molecular details of how intraluminal pH is sensed and transmitted to the cytosol are not fully understood. Peptidylglycine α‐amidating mono‐oxygenase (PAM; EC 220.127.116.11), a secretory pathway membrane enzyme which shares similar topology with two V‐ATPase accessory proteins (Ac45 and prorenin receptor), has a pH‐sensitive luminal linker region. Immunofluorescence and sucrose gradient analysis of peptidergic cells (AtT‐20) identified distinct subcellular compartments exhibiting spatial co‐occurrence of PAM and V‐ATPase. In vitro binding assays demonstrated direct binding of the cytosolic domain of PAM to V1H. Blue native PAGE identified heterogeneous high‐molecular weight complexes of PAM and V‐ATPase. A PAM‐1 mutant (PAM‐1/H3A) with altered pH sensitivity had diminished ability to form high‐molecular weight complexes. In addition, V‐ATPase assembly status was altered in PAM‐1/H3A expressing cells. Our analysis of the secretory and endocytic pathways of peptidergic cells supports the hypothesis that PAM serves as a luminal pH‐sensor, regulating V‐ATPase action by altering its assembly status. - Journal of Cellular Physiology, EarlyView.