Alveolar macrophages are cell's important in immune defense and inflammation in the lung, and the coagulation system participates in these reactions. In earlier experiments, it was found that alveolar macrophages contain and produce tissue factor, the extrinsic clotting pathway activator. The present experiments explore possible production by alveolar macrophages of the sequence of the clotting proteins that interact to form thrombin following initiation of coagulation by tissue factor. In studies using alveolar macrophages purified from rabbits, factor V activity was not detected in cell preparations assayed directly after isolation. However, after short-term culture, we found generation and release of factor V activity by these cells, which was predominantly from subpopulations with densities of 1.060-1.068 g/ml, corresponding to intermediate stages of alveolar macrophage maturation. Cell viability and protein synthesis were required for generation of the activity as shown by inhibitory effects of cell lysis before culture and by effects observed after including puromycin in cultures with viable cells. The activity generated was characterized as factor V by demonstrating specific functional requirements in one- and two-stage coagulation tests. There was no detectable generation in these cultures of factors II, VII, X, or the more recently described factor X-independent monocyte/macrophage prothrombinases. Factor V activity generated by alveolar macrophages may contribute to prothrominase assembly, activation of clotting, and fibrin formation within the alveolus.