Spread monolayers containing hydrophobic pulmonary surfactant protein, SP-B or SP-C, or SP-B/SP-C (2:1, w/w), alone or mixed with dipalmitoylphosphatidylcholine (DPPC) or dipalmitoylphosphatidylglycerol (DPPG), were formed on saline subphases containing calcium ions. Surface pressure-area characteristics of the films of the proteins were not affected by the presence of Ca2+ in the subphase. Calcium ions did not alter the surface properties of the binary and ternary films of DPPC plus either SP-B, or SP-C, or SP-B/SP-C (2:1, w/w). Surface pressure-area isotherms for the spread films of DPPG plus hydrophobic surfactant protein were Ca(2+)-dependent. The exclusion pressures of SP-B, SP-C and SP-B/SP-C (2:1, w/w) from protein-DPPG films in the presence of calcium were lower than the exclusion pressures in the absence of Ca2+. The divalent cation appeared to suppress the ability of SP-C and SP-B/SP-C (2:1, w/w) to remove phospholipid during squeeze-out from their mixed films with DPPG. The effects of Ca2+ on the monolayers of DPPG plus hydrophobic surfactant proteins were consistent with calcium producing diminished lipid-protein interactions, possibly resulting from Ca(2+)-induced changes in the ionization state and molecular packing of DPPG.