In the non-inflamed lung, surfactant protein A (SP-A) acts as an anti-inflammatory molecule through its effects on macrophage (M) function, modulating cytokine and reactive oxygen and nitrogen intermediate production. The receptors responsible for these effects of SP-A on human M are not clear, although SP-A binding to several proteins has been described. In this study, we demonstrate high-affinity specific binding of SP-A to primary human MSP-A binding was inhibited by EGTA, indicating calcium-dependence. However, mannan did not inhibit SP-A binding, suggesting that binding is mediated by a direct protein-protein interaction that does not involve carbohydrate recognition. Our laboratory has previously shown that SP-A is rapidly endocytosed by human Minto discrete vesicles. Although previous work indicates that SP-A is ultimately degraded by murine M over time, the trafficking pathway of SP-A through M following uptake has not been reported, and is of potential biologic importance. We examined trafficking of SP-A in human M by electron and confocal microscopy, and show for the first time that SP-A is endocytosed by primary human M through clathrin-coated pits and co-localizes sequentially over time with the early endosome marker EEA1, late endosome marker lamp-1, and lysosome marker cathepsin D. We conclude that SP-A binds to receptor(s) on human M, is endocytosed by a receptor-mediated, clathrin-dependent process, and traffics through the endolysosomal pathway. These studies provide further insight into the interactions of SP-A with the M cell surface and intracellular compartments that play important roles in SP-A modulation of lung M biology.