The yeast Saccharomyces cerevisiae is a traditional biotechnological organism, which has been used for the production of different heterologous proteins on the industrial scale for the last three decades. Advantages of using S. cerevisiae for production of heterologous proteins are secretion of proteins into cultivation medium, and growth on simple substrates and under undemanding cultivation conditions. The aim of the study was to test different signal sequences for secretion of recombinant phytase into the culture medium. We constructed plasmids with a constitutive promotor PTEF1 and inducible promoter PGAL1. The plasmids were transformed into S. cerevisiae INVSc1, cultivated in the synthetic medium without uracil and with glucose (SC-URA + GLU). We compared extracellular and intracellular phytase activity of clones with these promoters. We prepared plasmids with a constitutive promoter PTEF1 and with signal sequences of four different genes (MFα1, PLB1, CRH1, AGA2). We measured extracellular and intracellular phytase activity in YPD rich medium and in SC-URA + GLU medium at 28 °C and 37 °C. We showed that the type of medium affects the measured extracellular phytase activity. Increased cultivation temperature did not affect the extracellular phytase activity. We found that the signal sequence of the gene MFα1 in the YPD medium has the highest extracellular phytase activity and the signal sequence of the gene AGA2 has the lowest extracellular phytase activity.