To study an endocytotic role of the GTP-binding protein RhoA in Xenopus oocytes, we have monitored changes in the surface expression of sodium pumps, the surface area of the oocyte and the uptake of the fluid-phase marker inulin. Xenopus oocytes possess intracellular sodium pumps that are continuously exchanged for surface sodium pumps by constitutive endo- and exocytosis. Injection of Clostridium botulinum C3 exoenzyme, which inactivates Rho by ADP-ribosylation, induced a redistribution of virtually all intracellular sodium pumps to the plasma membrane and increased the surface area of the oocytes. The identical effects were caused by injection of ADP-ribosylated recombinant RhoA into oocytes. The C3 exoenzyme acts by blocking constitutive endocytosis in oocytes, as determined using a mAb to the beta 1 subunit of the mouse sodium pump as a reporter molecule and oocytes expressing heterologous sodium pumps. In contrast, an increase in endocytosis and a decrease in the surface area was induced by injection of recombinant Val14-RhoA protein or Val14-rhoA cRNA. PMA stimulated sodium pump endocytosis, an effect that was blocked by a specific inhibitor of protein kinase C (Gö 16) or by ADP-ribosylation of Rho by C3. Similarly, the phorbol ester-induced increase in fluid-phase endocytosis in oocytes was inhibited by Gö 16, C3 transferase, or by injection of ADP-ribosylated RhoA. In contrast to C3 transferase, C. botulinum C2 transferase, which ADP-ribosylates actin, had no effect on sodium pump endocytosis or PMA-stimulated fluid-phase endocytosis. The data suggests that RhoA is an essential component of a presumably clathrin-independent endocytic pathway in Xenopus oocytes which can be regulated by protein kinase C.