The literature contains variable reports concerning the hydrolysis of esculin by members of the family Enterobacteriaceae and particularly Escherichia coli. We examined 113 strains of fresh clinical isolates of E. coli and assessed the ability of colonies in a population to hydrolyze esculin with and without preincubation in inducible substrates at 24, 48, and 72 h. The number of strains capable of fermenting salicin, a sugar with a beta-glucoside linkage like esculin, was studied under the same conditions. A strip test that measured the presence of the constitutive glucosidase was also performed with and without preincubation in inducible substrates. No E. coli strain was able to produce constitutive enzyme; preincubation in esculin and salicin resulted in an induction of the beta-glucosidase. The number of colonies able to hydrolyze esculin increased with time. Only those strains preincubated in esculin or salicin were able to produce a positive constitutive strip test. Because the beta-glucosidase of E. coli is inducible, one should employe, when using growth media, a light inoculum obtained by touching the top of a colony with a bacteriological wire and read the reaction between 18 and 24 h, or perform a rapid strip or spot test.