The heterotrimeric GTP-binding regulatory proteins (or G proteins) play an important role in regulation of membrane signal transduction. Previously, we have identified the beta-subunit of G proteins (G beta) to be closely associated with mitotic spindles. In the present study, we have conducted experiments to examine whether the G alpha subunit is also associated with mitotic spindles.We prepared polyclonal antibodies against synthetic oligopeptides from different G alpha subunits and used them to localize G alpha in a nasopharyngeal carcinoma cell line by both immunohistochemistry and immunoelectron microscopy, and to see whether any specific G alpha subunit was present in the mitotic spindle region. Subsequently, we used a colocalization method to identify the overlapping distributions of specific G alpha subunits and beta-tubulin. To be sure that the findings represented a general phenomenon, several other cell types were also examined. To identify whether the G alpha subunit in the mitotic spindle was present in a heterotrimeric form, we performed ADP ribosylation experiments.(a) Only Go alpha subunit but not Gi alpha or Gs alpha was localized in the mitotic spindle. (b) Go alpha was colocalized with beta-tubulin in the mitotic spindle. (c) The precise site of localization of Go alpha on spindle microtubules was confirmed by immunoultrastructural study. (d) The association of Go alpha with the mitotic spindle could be found in both human and animal cell types. (e) The mitotic spindle preparation could be ADP ribosylated in the presence of pertussis toxin.The heterotrimeric Go protein is shown to be closely associated with the mitotic spindle. This finding suggests that Go protein may play some role in the regulation of mitotic spindles in addition to its regulation of signal transduction in neuronal cell membranes.