TRAIL (APO-2 ligand) and CD95L (CD95/APO-1/Fas ligand) share the highest homology among the TNF family members and the ability to induce apoptosis. These similarities raise the issue of a potential functional redundancy between the two ligands. We have previously shown that CD95L-resistant cells may be sensitive to TRAIL, even though apoptosis induced by both ligands is blocked by caspase inhibitors. Here we investigated TRAIL protein expression in cells of T and B origin and compared its regulation of expression with that of CD95L. A rabbit antibody (Ab) to a peptide sequence in the extracellular region of TRAIL identified recombinant TRAIL (rTRAIL) produced by Sf9 cells as a protein of approximately 32-33 kDa and soluble rTRAIL as a 19-20-kDa protein. In human and mouse cells, the Ab identified a 33-34-kDa and an additional 19-20-kDa protein only in human cells. Both transformed cells of the T and B lymphocyte lineage were found to react with the anti-TRAIL Ab by immunoblot analysis and surface staining. The majority of the cells analyzed co-expressed TRAIL and CD95L. Two cell lines showed a mirror-pattern, one being TRAILhigh CD95Llow and the other TRAILlow CD95Lhigh, thus suggesting the existence of a cell type-specific regulation of expression of the two ligands. Differently from CD95L, surface TRAIL was not up-regulated by any of the metalloprotease inhibitors tested, independently of the cell type analyzed. Conversely, reactivity with the anti-TRAIL but not with the anti-CD95L Ab was enhanced by cysteine protease inhibitors. An in vitro cleavage assay showed that generation of soluble rTRAIL was dependent on the functional activity of cysteine proteases, as it was blocked by leupeptin and E64 but not by the metalloprotease inhibitor 1,10-phenanthroline. Thus, even though TRAIL and CD95L share structural and functional properties, they have unique properties as they differ in their regulatory pathways, i.e. cell-type-dependent expression and sensitivity to protease inhibitors.