In order to explore the role of certain GTP binding proteins in the rat anterior pituitary, we have analyzed the subcellular distribution of the proteins rho and rab. They were found in both membrane and cytosolic fractions. Rab1 and rab2 were localized in both Golgi and endoplasmic reticulum (ER) membranes, while rab4 and rab6 were found in fractions enriched with Golgi and plasma membranes, implicating these proteins in the control of vesicular intracellular trafficking as described in other systems. Rab3 was localized like a fraction of synaptophysin, suggesting a role for rab3 in the targeting of "synaptic-like' microvesicles. We have identified three substrates of C. botulinum exoenzyme C3. A 26-kDa substrate with an isoelectric point (pI) of 5.2, probably rhoB, was localized in the lightest fractions such as rab3 and synaptophysin proteins. Two other 23-24 kDa substrates with pI of 5.5-5.8, probably rhoA and/or rhoC, were found in both fractions enriched with ER and secretory granules. Rho proteins have been implicated in the control of actin polymerization. Their localization in anterior pituitary suggests that rhoB could control the association of synaptic-like microvesicles and plasma membrane, and that rhoA/rhoC could play a role in secretory granule exocytosis; these two pathways being involved in cytoskeleton protein reorganisation in response to extracellular signals.