Pulmonary surfactant protein A (SP-A) is synthesized by type II cells and stored intracellularly in secretory granules (lamellar bodies) together with surfactant lipids and hydrophobic surfactant proteins B and C (SP-B and SP-C). We asked whether the progressive decrease in pH along the exocytic pathway could influence the secondary structure and lipid binding and aggregation properties of porcine SP-A. Conformational analysis from CD spectra of SP-A at various pH values indicated that the percentage of alpha-helix progressively decreased and that of beta-sheet increased as the pH was reduced. The protein underwent a marked self-aggregation at mildly acidic pH in the presence of Ca2+, conditions thought to resemble those existing in the trans-Golgi network. Protein aggregation was greater as the pH was reduced. We also found that both neutral and acidic vesicles either with or without SP-B or SP-C bound to SP-A at acidic pH as demonstrated by co-migration during centrifugation. However, the binding of acidic but not neutral vesicles to SP-A led to 1) a striking change in the CD spectra of the protein, which was interpreted as a decrease of the level of SP-A self-aggregation, and 2) a protection of the protein from endoproteinase Glu-C degradation at pH 4.5. SP-A massively aggregated acidic vesicles but poorly aggregated neutral vesicles at acidic pH. Aggregation of dipalmitoylphosphatidylcholine (DPPC) vesicles either with or without SP-B and/or SP-C strongly depended on pH, being progressively decreased as the pH was reduced and markedly increased when pH was shifted back to 7.0. At the pH of lamellar bodies, SP-A-induced aggregation of DPPC vesicles containing SP-B or a mixture of SP-B and SP-C was very low, although SP-A bound to these vesicles. These results indicate that 1) DPPC binding and DPPC aggregation are different phenomena that probably have different SP-A structural requirements and 2) aggregation of membranes induced by SP-A at acidic pH is critically dependent on the presence of acidic phospholipids, which affect protein structure, probably preventing the formation of large aggregates of protein.