This paper presents a new method, steady-state regulatory flux balance analysis (SR-FBA), for predicting gene expression and metabolic fluxes in a large-scale integrated metabolic–regulatory model. Using SR-FBA to study the metabolism of Escherichia coli, we quantify the extent to which the different levels of metabolic and transcriptional regulatory constraints determine metabolic behavior: metabolic constraints determine the flux activity state of 45–51% of metabolic genes, depending on the growth media, whereas transcription regulation determines the flux activity state of 13–20% of the genes. A considerable number of 36 genes are redundantly expressed, that is, they are expressed even though the fluxes of their associated reactions are zero, indicating that they are not optimally tuned for cellular flux demands. The undetermined state of the remaining ∼30% of the genes suggests that they may represent metabolic variability within a given growth medium. Overall, SR-FBA enables one to address a host of new questions concerning the interplay between regulation and metabolism.