We have recently described a putative receptor for lung surfactant protein-A (SP-A) on rat type II pneumocytes. The receptor, P63, is a 63 kDa type II transmembrane protein. Co-incubation of type II cells with P63 antibody (Ab) reversed the inhibitory effect of SP-A on secretagogue-stimulated surfactant secretion from type II cells. To further characterize SP-A interactions with P63, we expressed recombinant P63 protein in E. coli and generated antibodies to P63. Immuno-gold electron microscopy confirmed endoplasmic reticulum and plasma membrane localization of P63 in type II cells with prominent labeling of microvilli. Binding characteristics of iodinated SP-A to type II cells in the presence of P63 antibody were determined. Binding (4°C, 1h) of ¹²⁵I-SP-A to type II cells demonstrated both specific (calcium-dependent) and non-specific (calcium-independent) components. Antibody to P63 protein blocked the specific binding of ¹²⁵I-SP-A to type II cells and did not change the nonspecific SP-A association. A549 cells, a pneumocyte model cell line, expressed substantial levels of P63 and demonstrated specific binding of ¹²⁵I-SP-A that was inhibited by anti-P63 Ab. The secretagogue (cAMP)-stimulated increase in calcium-dependent binding of SP-A to type II cells was blocked by the presence of P63 Ab. Transfection of type II cells with siRNA to P63 reduced P63 protein expression, attenuated P63-specific SP-A binding and reversed the ability of SP-A to prevent surfactant secretion from the cells. Our results further substantiate the role of P63 as an SP-A receptor protein localized on the surface of lung type II cells.