P type ATPases have been identified in most of the studied organisms, where they catalyze ATP-dependent ion transport through cellular membranes and are able to form a phosphorylated intermediate during their catalytic cycle. These pumps transport a variety of substrates such as H⁺, Ca²⁺, Na⁺, K⁺, Mn²⁺, Cu²⁺, Cd²⁺ and aminophospholipids. Among the P-type ATPases, the members of the P5 subfamily still have their substrate specificity unassigned. These ATPases are found in all eukaryotic genomes analyzed to date and present a number of striking features that set them apart from the other enzymes of this type. There are two P5 ATPases encoded by Saccharomyces cerevisiae genome, namely YOR291w and YEL031w or SPF1. Spf1 ATPase shares 50% identity with Cta4 ATPase of the fission yeast Schizosaccharomyces pombe. It is known that cta4⁺ gene codifies for an ATPase located in the endoplasmic reticulum, which is involved in calcium homeostasis. To elucidate the physiological role of P5 ATPases and characterize SPF1, we analyzed (i) the SPF1 gene disruption and (ii) the over-expression of Cta4 ATPase in spf1 mutant. Yeast P-type ATPase mutants commonly display phenotypic defects in response to variations in the concentration of cations in the growth media. Consistent with this idea, we found that the growth of spf1 mutant cells was inhibited by 200 mM of CaCl₂. Furthermore, determination of ⁴⁵Ca²⁺ transport in cellular membranes isolated from wild type and spf1 cells revealed that the loss of SPF1 resulted in 2.5-fold decrease in ATP-dependent FCCP-insensitive ⁴⁵Ca²⁺ transport in total membranes which was restored to wild-type levels after exposure of spf1 cells to high extracellular Ca²⁺. Examination of calcium influx in yeast cells mediated by calcium channels as well as its regulation by calcineurin revealed a further difference between wild-type and spf1 cells. Cells lacking SPF1 was less effective than wild-type cells to induce Ca²⁺ uptake in response to high Ca²⁺ stress. Unlike wild-type cells under Ca²⁺ stress conditions, spf1 cells exhibited only weak stimulation of ⁴⁵Ca²⁺ uptake which was mainly insensitive to calcineurin inhibition by cyclosporine A. The data point to the crucial role of SPF1 in the Ca²⁺-ATPase and high-affinity Ca²⁺ influx system activities in yeast.