Castanospermine (CAST) is a known and potent inhibitor of various alpha-glucosidases in eukaryotes. In this work, we elucidated whether CAST could also be used for determining bacterial alpha-glucosidase activity, when measured with 4-methylumbelliferyl-alpha-D-glucoside as a substrate, both in a complex bacterial community, in activated sludge and in pure cultures of bacterial isolates. We found that 140 muM CAST inhibited alpha-glucosidase activity by 30% in a pure culture of Pseudomonas stutzeri. The a-glucosidase activity in Chryseobacterium gleum was inhibited by 90% at a concentration of 150 muM CAST, whereas the alpha-glucosidase in Paracoccus denitrificans was resistant to the inhibitor. CAST (140 muM) reduced alpha-glucosidase activity in activated sludge by 40%. the respiration rate being reduced by only 12%. No significant inhibition of the respiration rate was observed in Ps. stutzeri or Pa. denitrificans, whereas the respiration rate in C. gleum grown in a medium containing starch was inhibited by 50% with 140 muM CAST. No effect of CAST was observed in C. gleum grown in a complex medium. This indicated that CAST, at the concentration used, did not cause a general negative effect on bacterial activity. The results suggest that the CAST assay may potentially be useful in determining whether a-glucosidase activity, starch, poly- and disaccharides contribute appreciably to the overall activity of a bacterial community. However, the assay should not be used for quantitative measurements of such activity.