In Bacillus pasteurii glutamine is being taken up efficiently by a sodium-dependent uptake system and subsequently hydrolysed to ammonium and glutamate. Concerning the latter process, a catabolic L-glutamine amidohydrolase (glutaminase) was isolated from the cytoplasm of this alkaliphilic bacterium and purified to homogeneity using liquid chromatography. Biochemical and physical parameters of the pure enzyme were examined in detail. Interestingly, analysis of the glutaminase revealed a marked increase in catalytic activity in the presence of phosphate, a property yet restricted to animal glutaminases. This is the first report on the presence of a phosphate-activated glutaminase in bacteria.