A new substrate for the detection of bacterial beta-D-glucosidase was evaluated as an alternative to aesculin. This substrate, 3,4-cyclohexenoesculetin-7-beta-D-glucoside, was compared with aesculin for the detection of beta-D-glucosidase in 150 enterococci, 40 streptococci, 12 Listeria sp. and 250 strains of Enterobacteriaceae. In the Gram-positive strains tested, aesculin hydrolysis correlated with hydrolysis of 3,4-cyclohexenoesculetin-7-beta-D-glucoside. In the Gram-negative strains the new substrate was hydrolysed by all aesculin-positive strains and also by four strains (10%) of Escherichia coli which gave a negative aesculin reaction. 3,4-Cyclohexenoesculetin-7-beta-D-glucoside was shown to be a reliable alternative to aesculin and was shown to have significant advantages over aesculin when incorporated into solid media. This was due to the non-diffusible end product produced by hydrolysis of 3,4-cyclohexenoesculetin-7-beta-D-glucoside in the presence of iron.