The addition of several kinds of fatty acid to the culture medium of a recombinant human serum albumin (rHSA)-producing yeast, Pichia pastoris, resulted in increased expression levels of the product. Among the fatty acids tested, a small amount of oleic acid (0.01% (w/v)) doubled the rHSA production level in a shake-flask culture when measured by the reversed passive hemagglutination assay method. To elucidate this phenomenon, studies were conducted using deletion mutants from the AOX2 promoter region. Deletion mutants, designed for a detailed evaluation of the methanol regulation elements (AOX2-UAS, AOX2-URS1, and AOX2-URS2) did not respond to the addition of oleic acid. However, a deletion mutant that was not lacking an upstream region from the AOX2 promoter showed a response to oleic acid. The results implied the presence of an oleic acid-responsive element between nucleotides (nt) - 1529 and -803, and it may lie between nt - 1411 and - 1403 in the AOX2 promoter of P. pastoris. The response to oleic acid was shown to function even when the level of rHSA expression was increased by a mutation in the AOX2 promoter. Therefore addition of oleic acid to the medium Is likely to play an important role, in cooperation with gene manipulation, in achieving high expression levels of rHSA for the purpose of commercial production.