Recombinant barley alpha-amylase isozymes 1 and 2 were secreted by Pichia pastoris at up to 50 and 1 mg/liter, respectively, representing approximately a 50-fold increase compared to the levels of the heterologous expression by Saccharomyces cerevisiae. The cDNA clones E or pM/C encoding isozymes 1 and 2, respectively, were placed under the control of regulatory sequences from the Pichia AOX1 gene in the vector pHIL-D2. Both isozymes were effectively secreted to the medium as directed by their own signal sequences and easily purified to homogeneity in quantitative yield by affinity chromatography on beta-cyclodextrin-Sepharose. The N-terminal sequence, pI, and Mr indicated that native-like processing took place. Electrospray ionization mass spectrometry, however, revealed microheterogeneity for recombinant isozyme 1. While Mr of one recombinant isozyme 1 form of 45,452 was in excellent agreement with a value of 45,447 calculated from the sequence, liquid chromatography/mass spectrometry of endo Lys C-generated peptides followed by tandem mass spectrometry on a nanoelectrospray ionization/mass spectrometry/mass spectrometry system identified additional recombinant isozyme 1 forms to be glycosylated on Thr410, N-acetylated on His1, S-glutathionylated on Cys95, or C-terminally truncated of -412RS, -411QRS, and -410LQRS. The recombinant enzymes and the alpha-amylases from barley malt closely resembled each other in enzymatic activity on insoluble Blue Starch, amylose of degree of polymerization 17, and 2-chloro-4-nitrophenyl beta-D-maltoheptaoside as well as in Ca2+ dependency of activity. Pichia pastoris thus produced in high yields recombinant alpha-amylase that is similar with respect to structure and function to the enzyme purified from malt extracts. This greatly facilitates future mutational analysis of barley alpha-amylase in order to probe structure/function relationships.