A new acid-labile alpha-amylase (A-3) was isolated and purified from Aspergillus kawachii in barley-koji by ion-exchange chromatography followed by gel-filtration chromatography, and changes in its level during the production of shochu-koji, together with changes in the levels of two known acid-stable alpha-amylases (A-1 and A-2) in the same shochu-koji, were studied. SDS-PAGE analysis gave the molecular weight of A-3 as 56,000. Amino terminal analysis by a protein sequencer provided the sequence of the first fifteen residues as Ala-Thr-Pro-Ala-Asp-Tyr-Arg-Leu-Gln-Ala-Ile-Tyr-Phe-Leu-Leu. At 40-degrees-C, the formation of A-3 could be constant by detected up to 54 h. In the conventional method of shochu-koji making, the formation of A-3 cannot be detected after 36 h. A-3 was preferentially formed (up to 36 h) in the presence of 2% (w/w barley) citric acid. The results suggest that the formation of A-3 is affected not only by the temperature but also by the initial concentration of citric acid in the barley.