Surfactant subtypes have been identified by gradient centrifugation and characterized by differences in protein composition, vesicle size, and surface activity. The presence of surfactant proteins in each subtype is thought to be critical to the formation of aggregates and their function (Baritussio, Am J Physiol 1994;266:L436). To evaluate whether synthetic surfactant peptides interact with lipids in a manner comparable to native surfactant proteins, we prepared surfactant vesicles containing synthetic 125I-SP-B1-78 and phospholipids (DPPC:PG:Palmitic acid, 7:2:1) [PL], with and without palmitoylated synthetic SP-C1-34. We separated the aggregates by continuous sucrose gradient (0.1-0.8 M; 100,000 g for 48 h), collecting 50 fractions, and analyzed the PL and peptide profile. The PL peak appeared in fraction 17 and > 80% of PL was recovered in fractions 15-17, at a density of 1.07 g/mL, irrespective of the presence of SP-C. In the SP-B/PL aggregates, SP-B peaked at 51% in fraction 15 and > 95% of SP-B appeared in fractions 11-17. In the SP-B&SP-C/PL aggregates, 17% of the SP-B was detected at the bottom of the gradient with <5% PL and ≈78% was recovered in fractions 5-15 with a peak of 28% in fraction 13. In both aggregates, small amounts of PL, but no SP-B, were recovered at the top of the gradient, at a density of about 1.010 g/mL. In summary, synthetic SP-B associates with phospholipid aggregates at a density of about 1.07 g/mL, as shown by the overlapping peaks. The presence of SP-C does not affect the phospholipid distribution, but changes the SP-B profile significantly. These data are consistent with those obtained for term rabbit surfactant and suggest that the insertion of synthetic surfactant proteins in a simplified phospholipid dispersion reproduces important characteristics of native surfactant.