The effects of cytochalasin B (CB) on pancreatic structure and amylase release were studied by use of pancreatic fragments, isolated acini and isolated acinar cells. In pancreatic fragments and isolated acini CB caused the disappearance of microfilaments underlying the apical plasma membrane, loss of apical microvilli and luminal swelling, the last of which was greatly enhanced by addition of protein secretagogues. CB had no effect on basal amylase release but inhibited bethanechol-stimulated amylase in both fragments and acini. Isolated acinar cells, while retaining overall polarity, had lost most of the apical specialization including the microfilament and microvillous complex. Cells were still able to release amylase in response to bethanechol but this release was not affected by CB. The only structural effect of CB on isolated cells was margination of zymogen granules against the plasma membrane. This was, however, not accompanied by increased amylase release. It is concluded that microfilaments are important in maintaining the pancreatic acinar structure. Interference with this structure by CB leads to inhibition of bethanechol-stimulated amylase release. Microfilaments, however, may not play a direct role in secretion.