[(4-methoxy-4(3-beta-d-galactose-4-chlorophenyl)]spiro[1,2-dioxetane-3-1 ,3-tricyclo[7.3.1.0(2,7)]tridec-2,7-ene] ("s beta-Gal 102") and sodium [4-methoxy-4(3-beta-d-glucuronic acid-4-chlorophenyl)]spiro[1,2-dioxetane-3-1,3-tricyclo[7.3.1.0(2,7)]tri dec-2,7-ene] ("s beta-Glucor 102") are carbohydrate-containing 1,2-dioxetane compounds that produce chemiluminescence upon enzymatic hydrolysis by beta-d-galactosidase, and beta-d-glucuronidase, respectively. In this study, we have characterized and validated a sensitive detection principle for viable Escherichia coli based on enzymatic cleavage of s beta-Gal 102 and s beta-Glucor 102 ("ColiLight II"). The proposed chemiluminescent assay was optimized with respect to analytical requirements including incubation time, temperature, pH, enzyme induction, and cell permeabilization. The sensitivity and specificity rates of the assay were tested on ten different bacterial genera. The assay was found to be representative based on low coefficients of variations for both accuracy and precision. The analysis time was less than 1 h and the analytical detection limit was 10(2) to 10(3) E. coli cells. In combination with membrane filtration and a brief resuscitation step of 4 h, the proposed assay was capable of detecting low concentrations of stressed E. coli in potable water (< 30 CFU 100 ml(-1)). The proposed chemiluminescent enzyme assay may be used for assessing the metabolic activity of E. coli in oligotrophic environments and for early warning detection of low concentrations of E. coli in water for human consumption.