Carboxypeptidase E (CPE) is a prohormone-processing enzyme and peripheral membrane protein of endocrine/neuroendocrine secretory granules. CPE has been shown to bind to an amino-terminal peptide of pro-opiomelanocortin (N-POMC) at pH 5.5 and hypothesized to be critically involved in the targeting of hormones such as POMC to the regulated secretory pathway [Cool, D. R., Normant, E., Shen, F., Chen, H. C., Pannell, L., Zhang, Y., and Loh, Y. P. (1997) Cell 88, 73-83]. To further explore the possibility that CPE serves to mediate the association of content proteins with the membrane during granule biogenesis, the binding of CPE to granule content proteins was investigated using an in vitro aggregation assay in which the selective precipitation of granule content proteins is induced by titration of the pH to <6.0. CPE was observed to co-aggregate efficiently with pituitary and chromaffin granule content proteins at concentrations well below those that promote its self-aggregation. In addition, CPE co-precipitated at pH 5.8 with purified prolactin and with insulin, which homophillically self-aggregate yet are structurally distinct from N-POMC. N-POMC when added to the assays did not inhibit the aggregation of CPE with prolactin or insulin, indicating that these interactions do not involve a binding site for N-POMC. The data show that CPE interacts at acidic pH with a variety of different content proteins, resembling in this regard other granule membrane proteins. The results support the idea that co-aggregation of abundant membrane proteins with content proteins is an important general mechanism for the sorting and retention of secretory granule proteins during granule maturation.