The site(s) at which mammary tissue enzymatically cleaves rat (r) PRL, and the possibility that the cleaving activity is cathepsin D, were investigated using mass spectrometry and enzyme inhibitors. Cleavage of intact rPRL [22,566 atomic mass units (amu)] by either mammary gland-conditioned medium or cathepsin D (both at pH 3) reduced the mass of the molecule by 397 amu. Subsequent reduction of the large rPRL fragment cleaved by either method generated two fragments of 16,364 and 5808 amu. The mass of the smaller fragment is consistent with the report of Vick et al. (Biochim Biophys Acta 931: 196-204, 1987) that its amino-terminal residue is Ser149. These results indicate that both enzyme preparations cleave rPRL by excision of the tripeptide Leu-Val-Trp (mass = 397 amu) between residues 145 and 149. The ability of both enzyme preparations to cleave rPRL at pH3 was inhibited by pepstatin A but not by phenylmethane sulfonyl fluoride, and both preparations were essentially inactive at pH7. Accordingly, the PRL-cleaving activity of rat mammary tissue is probably cathepsin D.