Cytochalasins are cytoskeleton disrupters, and cytochalasin E has been reported to increase intestinal paracellular permeability. In this study, the cytochalasin E effect on galactose transport has been investigated. Ussing- type chamber experiments show an inhibitory effect of 20 μM cytochalasin E on unidirectional mucosal to serosal flux of galactose. On the contrary, the opposite unidirectional flux is not modified by the inhibitor. Results using intestinal everted sacs and rings confirm that galactose uptake by the tissue is diminished by cytochalasin E. The effect appears already after 5 min incubation, depends on cytochalasin E concentration, and does not occur in the absence of Na+. The inhibition is accompanied by an increase in the apparent K(m) of the active sugar transport (11.5 vs.15.8 mM) without significant change in the V(max) (10.6 vs. 9.1 μmol · g-1 wet weight · 5 min-1). Cytochalasin E does not modify either galactose uptake by brush border membrane vesicles or Na+-K+ ATPase activity in the enterocytes, indicating that the inhibitory effect on the Na+-dependent sugar transport cannot be explained as a direct effect on SGLT1 activity or as an indirect effect through the Na+-K+ ATPase. Thus, our results suggest that cytochalasin E decreases SGLT1 activity indirectly through cytoskeleton disruption.