Hsp70 chaperones assist a large variety of protein folding processes in the cell by transient association with short peptide segments of proteins. The substrate binding and release cycle is driven by the switching between the low affinity ATP bound state and the high affinity ADP bound state of Hsp70. Considerable progress has been made recently by the identification of in vivo substrates for the Escherichia coli homolog, DnaK, and the molecular mechanisms which govern the DnaK-substrate interactions. Here we review the processes that generate DnaK substrates in vivo and the properties of these substrates, and we describe insights gained from structural and kinetic analysis of DnaK-substrate interaction.