This study examined the degree of additivity of several physiologically relevant protein and nonprotein inhibitors in impairing the surface activity of whole and extracted calf lung surfactant (LS and CLSE) on a pulsating bubble apparatus at 37 degrees C. Inhibitors investigated were albumin, hemoglobin, C16:0 and C18:1 lysophosphatidylcholine (LPC), oleic acid (OA), palmitoleic acid (PA), arachidonic acid (AA), and mixed red blood cell membrane lipids (RBCML). In the absence of inhibitors, LS (0.5 mg/ml) and CLSE (0.75 mg/ml) reached minimum surface tensions < 1 mN/m within 5 min of bubble pulsation (20 cycles/min, 50% area compression). Each inhibitor acting alone was able to reduce the surface activity of LS and CLSE, either raising minimum surface tension or increasing the time course of surface tension lowering or both. Several combinations of inhibitors exhibited additivity in impairing LS or CLSE activity at a lower concentration in mixtures than when present alone (albumin plus either C16:0 LPC, C18:1 LPC, or RBCML; hemoglobin plus either C16:0 LPC, C18:1 LPC, RBCML, PA, OA, or AA). The degree of additivity, however, was typically small in terms of the magnitude of reduction in inhibitor concentration or the rise in minimum surface tension relative to the effects of the most severe single inhibitor present. Substantial synergy was not found for any of the combinations of protein and nonprotein inhibitors investigated. Mixtures of albumin with PA or AA actually had a reduced inhibitory effect on LS and CLSE activity compared with the free fatty acids alone, apparently because of albumin binding of these molecules. In all cases, the detrimental effects of mixed inhibitors on LS and CLSE activity were reversed at increased surfactant concentration. These results indicate that surfactant dysfunction in acute respiratory distress syndrome (ARDS) could be increased in severity by interactions between some inhibitory substances, but that supplementation with exogenous CLSE would be effective in reversing inactivation by the mixtures of blood proteins, membrane lipids, and fatty acids studied.