Escherichia coli 3000 XIII formed biofilms on the surface of polyvinylchloride coupons in a modified Robbins device. Bacteriophage T4D+ infected cells in the biofilm and replicated. It is commonly held that bacteriophage cannot infect surface-attached bacteria (biofilms) because such bacteria are protected by an exopolymeric matrix that binds macromolecules and prevents their diffusion into the biofilm. To our knowledge this is the first observation that a bacteriophage can infect and multiply within cells growing as a biofilm.