Proteins (IEF's 1120, 8118, 8120) sharing similarity to the bovine Rho GDP dissociation inhibitor (GDI) have been identified in the human two-dimensional-gel database of keratinocyte proteins. Molecular cloning of the corresponding cDNAs showed that IEF 8118 is the human homolog of bovine GDI while IEF 8120 is a distinct although related protein. All available information indicates the IEF 1120 is a derivative of IEF 8120. The cDNAs coding for IEF's 8118 and 8120 were recombined into vaccinia virus and expressed in differentiated human keratinocytes and their effect on the actin cytoskeleton was assessed by immunofluorescence using TRITC-phalloidin. The results showed that overexpression of both GDI proteins leads to rounding up of the cells and loss of stress fibers and focal contact sites. In addition, the cell to cell adhesion belts gradually disappeared, an effect that was particularly pronounced in infected cells overexpressing IEF 8120. Taken together, the results imply that Rho GDI's play a role in modulating the activity of the Rho proteins as their overexpression mimics phenotypic changes associated with the inactivation of these proteins.