SP-A is a lung-specific pulmonary surfactant-associated protein containing a calcium-dependent carbohydrate recognition domain and collagen-like sequence. The protein is a major component of the extracellular form of surfactant known as tubular myelin. SP-A is thought to influence the surface properties of surfactant lipids and regulate the turnover of extracellular surfactant through interaction with a specific cell-surface receptor. These properties of SP-A are dependent on the presence of calcium. We have estimated calcium binding parameters for SP-A from binding data obtained by equilibrium dialysis and gel permeation chromatography. Our results suggest that each SP-A monomer binds two to three calcium ions in conditions chosen as similar to those found in the alveolar lumen. The binding data are best fit to a model incorporating two calcium binding sites with different affinities. Studies with a fragment of SP-A generated by limited proteolysis suggest the higher affinity site for calcium is located in the noncollagenous carboxy-terminal end of SP-A. This region of SP-A contains a carbohydrate recognition domain homologous to other C-type lectins. The binding of calcium to this region of SP-A causes a conformational change as assessed by a small change in the intrinsic fluorescence spectrum and a marked change in the susceptibility to proteolysis. At physiological calcium concentrations, intact SP-A aggregates in a reversible fashion, a property that may be relevant to the formation of tubular myelin.