An alpha-amylase (E.C. 3.2.1.1.) secreted by Lactobacillus amylovorus was partially purified and characterized. This high-molecular-weight enzyme [Imam SH, Burgess-Cassler A, Cote GL, Gordon SH, Baker FL (1991) Curr Microbiol 22:365-370] was quantified with a clinical alpha-amylase assay adapted to a microplate format. It was isolated from concentrated cell-free culture medium by ammonium sulfate precipitation, ion exchange, and hydrophobic interaction chromatographies. The enzyme was not particularly thermostable, but like three other microbial alpha-amylases tested for comparison, was renaturable following treatment with SDS and heat. The pH optimum and pl were 5.5 +/- 0.5 and 5.0, respectively; its temperature optimum was 60-65-degrees-C, and the molecular weight on SDS gels was 140 +/- 10 kDa.