Production of the killer toxin of Pichia kluyveri 1002 was stimulated in the presence of yeast extract. In a minimal medium production was optimal at pH 3.8-4.0 and 22--25 degrees C. Addition of gelatin and nonionic detergents, like Brij-58 (polyoxyethylene 20 cetyl ether) and Triton-X-100, to this medium enhanced production significantly. The killer toxin was purified 140-fold by use of a stepwise ethanol precipitation and butyl Sepharose column chromatography. The purified killer toxin, which still contained some carbohydrates, appeared to be glycoprotein with a mol wt of about 19 000 and an isoelectric point of 4.3. It was stable between pH 2.5 and 4.7 and up to 40 degrees C.