The 70-kilodalton heat shock protein (hsp70) family members appear to be essential components in a number cellular protein-protein interactions. We report here on the characterization of a new functional region in hsp70, a calmodulin-binding site. We have identified a 21-amino-acid sequence within the hsp70 protein that contains a calmodulin-binding domain. The peptide formed a potential amphipathic alpha helix and bound calmodulin with high affinity. Comparison of amino acid homology of this calmodulin-binding sequence with analogous hsp70 sequences from other species showed a high degree of conservation.