The present results show that stimulation of Intestine 407 epithelial cells with LTD4 (Leukotriene D4) triggers a rapid activation of the pertussis-toxin-sensitive Gi3-protein and a simultaneous translocation of its alpha-subunits to a crude cytoskeletal fraction. The activation of G alpha i3, which was measured as the GTP/ GDP exchange ratio, peaked about 15 s after the addition of LTD4. Western blot analyses of subcellular fractions showed that G alpha i3-subunits accumulated in the cytoskeletal fraction and decreased in the membrane fraction, and the decrease was most marked 15 s after the exposure to LTD4. None of the other pertussis-toxin-sensitive G-proteins (Gi3-Z and G alpha 0) were activated or translocated upon stimulation with LTD4. This agonist was also found to reduce the GTP/GDP exchange ratio of Gi-proteins without affecting the subcellular distribution of its alpha-subunits. These findings imply that the Gi3-protein is the pertussis-toxin-sensitive G-protein previously found to mediate several downstream LTD4-stimulated signalling events. Furthermore, the translocation of G alpha i3-subunits to the cytoskeleton and the simultaneous inhibition of G3-proteins indicate that the cytoskeleton might participate in the signalling process of human epithelial cells.