The chaperonin GroEL has an essential role in mediating protein folding in the cytosol of Escherichia coli. Here we show that GroEL interacts strongly with a well-defined set of approximately 300 newly translated polypeptides, including essential components of the transcription/translation machinery and metabolic enzymes. About one third of these proteins are structurally unstable and repeatedly return to GroEL for conformational maintenance. GroEL substrates consist preferentially of two or more domains with alphabeta-folds, which contain alpha-helices and buried beta-sheets with extensive hydrophobic surfaces. These proteins are expected to fold slowly and be prone to aggregation. The hydrophobic binding regions of GroEL may be well adapted to interact with the non-native states of alphabeta-domain proteins.