In this study we report on a novel structural phenotype in Escherichia coli biofilms: cellular chain formation. Biofilm chaining in E. coli K-12 was found to occur primarily by clonal expansion, but was not due to filamentous growth. Rather, chain formation was the result of intercellular interactions facilitated by antigen 43 (Ag43), a self-associating autotransporter (SAAT) protein, which has previously been implicated in auto-aggregation and biofilm formation. Immunofluorescence microscopy suggested that Ag43 was concentrated at or near the cell poles, although when the antigen was highly overexpressed, a much more uniform distribution was seen. Immunofluorescence, microscopy also indicated that other parameters, including dimensional constraints (flow, growth alongside a surface), may also affect the final biofilm architecture. Moreover, chain formation was affected by other surface structures; type I fimbriae expression significantly reduced cellular chain formation, presumably by steric hindrance. Cellular chain formation did not appear to be specific to E coli K-12. Although many urinary tract infection (UTI) isolates were found to form rather homogeneous, flat biofilms, three isolates, including the prototypic asymptomatic bacteriuria strain, 83972, formed highly elaborate cellular chains during biofilm growth in human urine. Combined, these results illustrate the diversity of biofilm architectures that can be observed even within a single microbial species.