In order to see whether the stringent response was involved in biofilm formation, Escherichia coli DS291 (MG1655), and its isogenic relA spoT derivative were grown for 48 h in a chemostat at dilution rates of 0.025 and 0.25 h(-1) under serine limitation. The absence of the stringent response genes relA and spoT had little effect on the planktonic cell concentrations. However, a significant (P < 0.001) reduction in biofilm cell density of the relA spoT mutants was seen at a doubling time of 40 h. At a doubling time of 4 h, differences in biofilm cell density were not significant. Scanning confocal laser microscopy demonstrated the cell densities of microcolonies in the relA spoT mutant to be lower than those in the wild type. Using a microtiter plate assay, we found biofilm formation in relA spoT mutants to be similarly reduced in minimal media but to be enhanced in rich media (Luria-Bertani broth). No significant differences in biofilm formation were observed between wild type and isogenic relA mutants under any growth conditions. Overall, these results suggest that both stringent response genes relA and spoT are important in nutrient-limited biofilms.