Phalloidin, a potent microfilament toxin, induces polymerization of actin in vitro and in vivo. In a permeability assay, bovine aortic endothelial cell cultured on microcarrier beads exclude significantly more serum albumin after 30 min treatment with 10(-6), 10(-8), and 10(-10) M phalloidin than controls. Furthermore, pretreatment of microcarriers with 10(-8) M phalloidin significantly reduces permeability increases by histamine, bradykinin, thromboxane A2 mimetic, and cytochalasin B, (all at 10(-6) M). Phalloidin also causes significant surface area and perimeter increases in cultured endothelial cells. The cells also display increased acting stress fibers and show a weblike cytoskeletal pattern of microfilaments. These data suggest that in vitro the endothelial junctional barrier may be enhanced in part by assembly of actin filaments.