The growing body of evidence points to an important role of the H⁺ and Ca²⁺ gradients in the protein targeting and the intracellular transport of membrane vesicles, however, the molecular bases of the formation of those gradients and their participation in these key processes of the cell biology remain obscure. Our lab has previously shown that the main organelles of the yeast secretory pathway such as ER, Golgi and probably nuclear envelope beside the vacuoles possess the V H⁺-ATPases, Ca²⁺-ATPases and Ca²⁺/H⁺ exchangers which create the H⁺ and Ca²⁺ gradients across respective membranes and participate in the homeostasis of those ions. However, it is still open to question: i) whether those transporters are present in the late secretory vesicles (SV) carrying macromolecules to the plasma membrane (PM) and cell wall and ii) whether PM has its own Ca²⁺-ATPase and/or Ca²⁺/H⁺ exchanger involved in ion homeostasis. In this work we used sec6-4 mutant (S. cerevisiae EHY 227) isolated from wild type strain S. cerevisiae EHY 239 expecting that if PM has own Ca²⁺ transporters the accumulation of SV at non permissive temperature (37^o) should result to the increase of the activity of the transporters in the region of light membranes. After the accumulation of SV at 37 ^oC total membranes were isolated from spheroplasts and fractionated in sucrose density gradient. As was expected the H⁺-translocase activity of P H⁺-ATPase has indeed moved from its typical location in high density membranes to the Golgi enriched lighter membranes. This finding is in a good agreement with the accumulation of the SV. Moreover, the highest peak of the Ca²⁺-ATPase activity and a high peak of the Ca²⁺/H⁺ exchanger were detected in the even more light membranes indicating that both Ca²⁺ transporters are good candidates to be PM enzymes. We also found that at least five distinct populations of SV were equipped with the V H⁺-ATPase, Ca²⁺-ATPase and Ca²⁺/H⁺ exchanger suggesting that all these ion transporters are important for the correct protein transport to the PM and the interaction of the SV with PM. Future experiments should be undertaken to unveil how these transporters and ion gradients participate in key processes in secretory pathway.