A proteolytic activity has been shown to be associated with the heat shock protein 70 (hsp70). In order to study this, I have constructed RNA transcribing vectors with the coding sequences of the D. melanogaster (pBUG7) and the human (pMAN70) genes coding hsp70, and with an internal deletion (pBUG301) in D. melanogaster. Proteins from 37 kDa to 70 kDa were translated in a rabbit reticulocyte lysate in the presence of 35S-methionine from RNA synthesized in vitro off the full length templates (pBUG7, and pMAN70), or altered templates. Restriction digestion of pBUG7 with BamH I and Nar I yields templates that produce carboxy-terminal truncated proteins of 37 kDa and 61 kDa respectively. The full length and the truncated proteins contain a proteolytic activity when assayed by SDS/PAGE in two dimensions. The internally deleted protein does not maintain the proteolytic activity. The proteolytic activity was shown not to be the result of non-enzymatic cleavage. A general serine proteinase inhibitor eliminates the proteolytic activity of the full length human and D. melanogaster hsp70. This evidence shows that the proteolytic activity is directly connected to hsp70.