Pulmonary surfactant protein A (SP-A) contains 4 domains: a disulfide forming amino terminus, a collagen-like region, a neck region, and a carbohydrate recognition region. The protein binds the lipids dipalmitoylphosphatidylcholine and galactosylceramide and induces aggregation of phospholipid vesicles. SP-A also inhibits lipid secretion and enhances the uptake of phospholipid by alveolar type II cells. Previously described monoclonal antibody 1D6 blocks the inhibitory effect of SP-A on lipid secretion by type II cells, but antibody 6E3 has no effect. In the present study we mapped the epitopes for monoclonal antibodies 1D6 and 6E3 by enzyme-linked immunoassay of recombinant proteins expressed using the baculovirus system, and investigated the domain that is responsible for the SP-A interactions with lipid. Monoclonal antibody 1D6 bound to mutant SP-As in which the neck portion of the molecule was deleted or substituted with that of mannose-binding protein A, but 6E3 failed to bind to these mutants. In contrast, 1D6 did not bind to a chimera in which the carbohydrate recognition domain (CRD) was substituted with that of surfactant protein D (SP-D). In addition, 1D6 failed to recognize antigen in cells infected with the recombinant virus directing the synthesis of a Cys204-Cys218 (small disulfide loop) deletion within the CRD. Antibody 1D6 completely blocked the binding of SP-A to dipalmitoylphosphatidylcholine and galactosylceramide and liposome aggregation. By comparison, 6E3 failed to completely attenuate the interactions of SP-A with lipids. However, both 6E3 and 1D6 blocked the uptake of lipid by type II cells that is caused by SP-A. From these data, we conclude that: 1) the epitope for antibody 6E3 is located at the neck domain of SP-A and that for antibody 1D6 is at the small loop region in the CRD; 2) the CRD is essential for the SP-A functions of lipid binding, liposome aggregation, the inhibitory effect on lipid secretion, and the augmentation of lipid uptake by type II cells, and these activities are largely attributable to amino acid residues within the steric inhibitory footprint of 1D6 bound to the small disulfide loop region; and 3) the neck domain of SP-A may also be involved in the process of SP-A-mediated uptake of phospholipids by alveolar type II cells.