Trypsin inhibitors in serum-free conditioned media (SFCM) of various human carcinoma cell lines were analyzed by reverse zymography. Most of the cells secreted high-molecular-weight trypsin inhibitors (HMTI) larger than 100 kDa. The cell lines of colorectal carcinoma origin had a tendency to secrete HMTI whose molecular weight was a little higher than that of the other cell lines. Analysis of SFCM of subclones with different histological differentiation and metastatic/invasive potentials derived from a single pancreatic carcinoma cell line SUIT-2 showed that the HMTI activity in SFCM was correlated to the degree of histological differentiation in vivo and tended to be inversely correlated to their metastatic/invasive capabilities. Immunoblotting analysis revealed that these HMTI were protease nexin-II/amyloid beta protein precursors (PN-II/APP). Semi-quantificative reverse-transcriptase/polymerase-chain reaction study for PN-II/APP mRNAs suggested that the differences in PN-II/APP activities in SFCM between the subclones might be post-transcriptional or post-secretional events. In addition, SFCM of a highly metastatic subclone contained 43-kDa protein which reacted to anti-APP monoclonal antibody (MAb) suggesting that the subclone may have APP-degrading activity.