Long-chain free fatty acids (FFAs) in pulmonary bronchoalveolar lavage fluid (BALF) are antimicrobial agents that may participate in lung defenses. FFAs may also participate in synthetic and metabolic activities of bronchoalveolar lining cells. In evaluating the origins of FFAs, we found that rat triglyceride lipase activity was readily detectable in rat BALF. This activity appeared to be caused mainly by lipoprotein lipase (LPL), because it was inhibited by protamine, a high salt concentration, or specific anti-LPL antibody. LPL activity was detected in BALF from guinea pigs, humans, and rabbits, but rats had significantly more LPL activity than the other species. LPL activity in rat BALF was enhanced by heat-inactivated serum, but LPL-mediated hydrolysis of triglycerides in BALF proceeded at 37 degrees C in vitro even without serum. The possibility that BALF contained an intrinsic LPL activating factor(s) was suggested by the fact that concentrated, heat-inactivated lavage was 85% as effective as heat-inactivated serum in enhancing the LPL activity of fresh BALF. Macrophages are the likely source of LPL in BALF, and we confirmed that rat resident alveolar macrophages produce LPL in culture in a time-dependent fashion. It was concluded that FFAs in BALF were produced by the hydrolysis of triglycerides by LPL.