A 15-mer phage display random peptide library was screened with purified bovine Hsc70, and nucleotide sequence analysis of the selected clones showed a large enrichment for peptides containing basic sequences with at least KK, KR, or RR. Binding affinity for Hsc70 of representative peptides increased dramatically for heptamers compared with hexamers. The peptide NIVRKKK had the highest affinity for Hsc70, and substitution analyses showed that hydrophobic residues followed by basic residues play important roles in maintaining this affinity. In contrast, NIVRKKK was a weaker stimulator of the Hsc70 ATPase activity compared with pigeon cytochrome c peptide and FYQLALT, a peptide optimized for binding to Hsc70. FYQLALT effectively blocked the binding of NIVRKKK to Hsc70, possibly by causing a conformational change that masked Hsc70's binding site for the basic peptide. Two hypotheses are offered to explain the two different peptide motifs. First, it is proposed that Hsc70 recognizes two different amino acid sequence motifs in its dual roles of chaperoning proteins to organelles (NIVRKKK-like sequences) and facilitating protein folding (FYQLALT-like sequences). Second, the NIVRKKK motif may be used to bind certain folded proteins with which Hsc70 interacts, such as itself, p53, and Dnaj2.