The secretion of toxic proteinaceous compounds is common phenomenon among yeast genera. Toxin properties are determined by natural habitats and depend on the extracellular conditions. We analyzed in details the effect of temperature and pH on the activity and stability of Saccharomyces cerevisiae K2 toxin. By performing large-scale and long-term analysis, pH value has been identified to be an essential factor for the acquisition of killer protein functionality. We have found that K2 killer toxin is produced in comparable levels at broad range of pH values, in clear contrast to activity profile of the protein. Protein, produced at basic pH conditions—incompatible with killer activity—can be recovered into active form by adjustment of pH to acidic values. Fully active protein is nevertheless sensitive toward pH shift: once established, activity is un-recoverably lost upon adjustment of pH to basic values. Acidic environment as an essential condition of maturation of active form of K2 killer protein is postulated.