The present results show that stimulation of Intestine 407 epithelial cells with LTD₄(Leukotriene D₄) triggers a rapid activation of the pertussis-toxin-sensitive G_i3-protein and a simultaneous translocation of its α-subunits to a crude cytoskeletal fraction. The activation of Gα_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 Gα_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 (G_i1-2and Gα₀) were activated or translocated upon stimulation with LTD₄. This agonist was also found to reduce the GTP/GDP exchange ratio of G_s-proteins without affecting the subcellular distribution of its α-subunits. These findings imply that the G_i3-protein is the pertussis-toxin-sensitive G-protein previously found to mediate several downstream LTD₄-stimulated signalling events. Furthermore, the translocation of Gα_i3-subunits to the cytoskeleton and the simultaneous inhibition of G_s-proteins indicate that the cytoskeleton might participate in the signalling process of human epithelial cells.