We have tested the effect of a preparation of rat lung lavage surfactant (SAM) on phagocytosis and intracellular killing of Staphylococcus aureus by rat alveolar macrophages (AMs). The SAM was isolated and purified by density gradient centrifugation. It was highly enriched in disaturated phosphatidylcholine, and invariably lowered the surface tension of a clean saline solution to < 10 dynes/cm at 37 ° C. We used a radiometric assay to measure phagocytosis as uptake by AMs of ¹⁴ C phenylalanine labelled staphylococci, and intracellular killing as incorporation of ³H thymidine by viable staphylococci from the lysed AMs. When staphylococci were incubated with 100 to 300 Μg of SAM, both phagocytosis and intracellular killing efficiency were enhanced. The mean numbers of intracellular staphylococci/AM were 24.3 ± 3.8 and 18.3 ± 3.0 in the SAM and control groups, respectively (p < 0.001). The mean intracellular killing efficiency was 59.6 ± 6.76% and 39.7 ± 7.17% in the SAM and control groups, respectively (p < 0.001). Previous studies have shown that unpurified rat lung lavage fluid enhances intracellular killing of staphylococci by AMs. Our results suggest that SAM may be the active principle in lung lavage fluid that enhances intracellular killing. In addition, in our test system, SAM enhanced staphylococcal phagocytosis by AMs.