In this report we have studied the morphological changes of the Golgi complex (GC) that specifically accompany F-actin reorganizations. In starved rat RBL-2H3 tumor mast cells, the GC, that was visualized at immunofluorescence level with antibodies raised against the Golgi-resident proteins giantin, mannosidase II, or TGN-38, showed a compacted morphology with a supranuclear positioning. Concomitant to membrane ruffle formation induced by epidermal growth factor (EGF) or phorbol 12-myristate 13-acetate (PMA), and stress fiber formation induced by lysophosphatidic acid (LPA), specific GC morphological changes were observed. When cells were stimulated with EGF or PMA, the compacted GC morphology was transformed into a reticular network that was extended towards the cell periphery. When cells were incubated with LPA, the GC acquired a characteristic ring-shaped morphology. Brefeldin A (BFA) did not affect the PMA- or LPA-induced membrane ruffling and stress fiber formation, respectively, indicating that actin rearrangements occurred independent of the presence of the GC. Upon BFA removal, the presence of PMA or LPA during the recovery process induced the GC to acquire the morphological appearance described above for each agent. Moreover, the PMA- but not the LPA-induced GC rearrangements were sensitive to the actin perturbing agents cytochalasin D and jasplakinolide. When cells were preincubated with the phosphatidylinositide 3-kinase (PI3K) inhibitors wortmannin or LY294002, the PMA-induced GC morphological changes were inhibited but not membrane ruffles. Finally, the PMA-induced increase in the post-Golgi transport of glycosaminoglycans to the cell surface was not altered by cytochalasin D or jasplakinolide. Altogether, these data suggest that: (1) the shape of the GC is influenced by the 3D arrangement of actin microfilaments; (2) PI3K regulates the association of the GC with actin microfilaments; and (3) actin microfilaments are not essential for the post-Golgi transport to the plasma membrane.