For use in protein folding studies, ribonuclease A (RNase A), a 124-residue protein with the C-terminal sequence Phe-Asp-Ala-Ser-Val, has been labeled site-specifically by a carboxypeptidase Y (CPase Y)-catalyzed transpeptidation reaction which replaces the C-terminal residue(s) of RNase A with the extrinsic fluorescent probe 3-(2-naphthyl)-L-alanine amide (Nal-NH2). It was found that this CPase Y-catalyzed transpeptidation required the presence of the chemical denaturant urea (ca. 5 M) and that, under these conditions, effective transpeptidation occurred only in the pH range of ca. 5.5 to 6.5. The two major products from this labeling reaction were purified to homogeneity by ion-exchange and reverse-phase chromatography and characterized by tryptic mapping, amino acid analysis, and mass spectrometry. The major product, obtained in the reproducible, isolated molar yield of 20%, is the derivative in which only the single C-terminal residue, Val-124, is replaced directly by the probe Nal-NH2. Another isolated product, obtained in 11% yield, is the derivative in which two of the C-terminal residues, Ser-123 and Val-124, are replaced by one Nal-NH2. Both of these purified derivatives are enzymatically active (85 and 18%, respectively), and both exhibit spectral properties characteristic of the extrinsic probe including an observed fluorescence lifetime which is approximately monoexponential (tau = 42 ns) when the naphthyl chromophore is excited in the region of its absorption band.