A tryptic fragment (residues 21-47) containing the signal peptide of ovalbumin has been isolated by reverse-phase chromatography. The peptide is more active at inhibiting the processing of pre-prolactin in an in vitro translation system than fragments of ovalbumin isolated previously, and is similar in properties to synthetic signal peptides. The ovalbumin signal fragment is shown to bind to a protein component of salt-stripped pancreatic microsomal membranes, which is cross-linked under UV irradiation by a radioactive synthetic photoaffinity signal peptide probe to yield a radiolabelled 45 kDa protein.