Two P-thymosins are expressed in most mammalian tissues. We detected small amounts of a third peptide in extracts of rabbit spleen. The portion of this peptide increased when the tissue was first frozen and then thawed at 4°C. Small amounts of the peptide are also present in cells from suspension cultures homogenized immediately in diluted perchloric acid. By means of amino acid analysis and MALDI-mass spectroscopy this peptide was identified to be a C-terminally truncated form of thymosin pio. Having studied the formation in more detail we found that after a 4-h thaw at 4°C all thymosin Pi 0 was truncated to thymosin Pio 1 41 5 which was further degraded during the next 20 h. On the other hand, thymosin p 4 Ala , the second P-thymosin being present in rabbit spleen, was not truncated or degraded even after 22 h. It might be possible that in vivo a truncated form of thymosin p 10 is formed by a carboxydipeptidase while thymosin P4 Ala is rather stable against proteolytic modification. By using a newly designed ultrafiltration assay, we determined the dissociation constants of the complexes of G-actin and these three P-thymosins to be 0.28, 0.72, and 0.94 nM for thymosin P4 Ala , Pio, and thymosin Pio 1 41 , respectively. The complex with p 4 Ala is unambiguously more stable than the complex with Pio or p 4 (0.81 nM). The change in the dissociation constant generated by the truncation of the two C-terminal amino acid residues of Pio is small but statistically significant. This demonstrates that even the very last amino acid residues at the C-terminus of P-thymosins are involved in the interaction with G-actin.© 1997 Federation of European Biochemical Societies.