A set of heptapeptides was used to compare the relative peptide affinities of three proteins of the hsp70 family: bacterial DnaK, mammalian cytosolic hsc70, and BiP from mammalian ER. Each hsp displays a characteristic pattern of relative affinities. DnaK and hsc70 are more similar to each other than to BiP. A difference in peptide binding specificity may be an important determinant in adjusting an hsp70 family member to its particular cellular function.