Viable Escherichia coli cells were entrapped in agar gel layers and incubated in a phosphate-limited glucose medium. Immobilized bacteria displayed enhanced alkaline phosphatase activity and overexpressed the outer membrane protein PhoE as compared with free-floating organisms. These observations highlighted the existence of high phosphate deprivation within biofilm-like structures. In addition, the antimicrobial efficacy of latamoxef against immobilized bacteria was partly recovered in the presence of a high phosphate concentration. From these data, a possible role of phosphate deprivation in the high resistance of sessile-like organisms to antibiotics may be considered.