High levels of soluble HLA-class4 molecules (sHLA) were found to be produced in a time-and dose-dependent manner by colon-adenocarcinoma Col0205 cells in response to IFN-y stimulation. Among other cytokines tested, only IL-6, TNF, IFN-a and IFN-p showed weak inducibility. IFN-y-induced production of sHLA was synergistically enhanced by IL-la, IL-6 or TNF, and combined treatment with TNF and IL-6 exhibited an additive to synergistic induction. Expression of sHLA is unlikely to result from IFN-y-induced enhancement of overall HLA-class4 expression, as at low concentrations IFN-a, IFN-P and IFN-y stimulated increased expression of cell membrane HLA-class4 molecules in Colo205 cells with almost equal efficiency, whereas only IFN-y induced high level production of sHLA. Immunoblotting with a monoclonal antibody recognizing p,-microglobulinfree HLA-class4 heavy chain revealed 3 major forms of sHLA heavy chain, i.e., 45143-, 37-and 33-kDa molecules, in the culture supernatants of IFN-y-induced Col0205 cells. The 45143-kDa proteins can be partitioned into Triton X-I 14, representing intact HLA-class4 heavy chains shed from the cell membrane. The hydrophilic 37and 33-kDa heavy chain, which remained almost exclusively in the aqueous phase after extraction with Triton X-l 14, could well be due to alternative RNA splicing, with deletion of exon 5 encoding the hydrophobic transmembrane region of membrane-anchored HLA-class4 heavy chain.