Chloroplast transit peptides have been proposed to function as substrates for Hsp70 molecular chaperones. Many models of chloroplast protein import depict Hsp70s as the translocation motors that drive protein import into the organelle, but to our knowledge, no direct evidence has demonstrated that transit peptides function either in vivo or in vitro as substrates for the chaperone. In this report, we demonstrate that DnaK binds SStp (the full-length transit peptide for the precursor to the small subunit of Rubisco) in vivo when fused to either glutathione-S-transferase (GST) or to an His₆-S-peptide tag (His-S) via an ATP-dependent mechanism. Three independent biophysical and biochemical assays confirm the ability of DnaK and SStp to interact in vitro. The cochaperones, DnaJ and GrpE, were also associated with the DnaK/SStp complex. Therefore, both GST-SStp and His-S–SStp can be used as affinity-tagged substrates to study prokaryotic chaperone/transit peptide interactions as well as to provide a novel functional probe to study the dynamics of DnaK/DnaJ/GrpE interactions in vivo. The combination of these results provides the first experimental support for a transit peptide–dependent interaction between a chloroplast precursor and Hsp70. These results are discussed in light of a general mechanism for protein translocation into chloroplasts and mitochondria.