Cytochalasins enhanced surfactant secretion from primary cultures of [³H]choline-labeled type II epithelial cells from the rat. Cytochalasins A, B, C, D and dihydrocytochalasin B enhanced secretion of phosphatidyl-[³H]choline ([³H]PC) in a dose-dependent manner with EC₅₀ values of 1, 2, 0.5, 0.1 and 1 μM for cytochalasins A, B, C, D and dihydrocytochalasin B, respectively. Only cytochalasin A caused significant cytotoxicity as determined by release of the intracellular enzyme lactate dehydrogenase (EC 1.1.1.17). Dose responses of surfactant release induced by cytochalasins B, C and D were biphasic; maximal release was observed between 0.1–1.0 μM for cytochalasins C and D between 1 and 10 μM for cytochalasin B. Secretion decreased toward control levels at concentrations of cytochalasin above these maximal concentrations. Increased rates of [³H]PC release were noted between 1 and 3 h after exposure to cytochalasin D. Increased rates of surfactant release induced by cytochalasin D were additive to release induced by the β-adrenergic agonist, terbutaline, or forskolin, although cytochalasin D had no direct effect on cytosolic cyclic AMP levels. Changes in cell shape and microfilament organization were observed by phase-contrast microscopy and fluorescence microscopy using rhodamine-conjugated phalloidin after exposure of the isolated type II cells to cytochalasin D. Disruption of microfilaments associated with lamellar bodies of the purified type II cells occurred after treatment with cytochalasin D. Cytochalasin D augmented surfactant release from purified type II cells and disrupted the microfilament structure of those cells, supporting the hypothesis that alterations in microfilaments are associated with surfactant release.