Interleukin-12 (IL-12) is unique amongst cytokines in being a disulfide-linked heterodimer of two separately encoded subunits (p35 and p40). We expressed single chain IL-12 proteins from retroviral constructs in which the two IL-12 subunits were linked by a 6-15 amino acid polypeptide linker, with deletion of the 22 amino acid leader sequence of the trailing subunit. The murine fusion protein IL-12.p40.L.delta p35 containing a (Gly4Ser)3 linker was stably expressed, bioactive in vitro, and had an apparent specific activity comparable to that of native and recombinant IL-12. Western blotting confirmed that murine IL-12.p40.L.delta p35 retained the linking polypeptide sequences. The analogous human IL-12.p40.L.delta p35 fusion protein containing a Gly6Ser linker was bioactive with an apparent specific activity comparable to recombinant human IL-12. In a preexisting CMS-5 tumor model, CMS-5 cells secreting either native or fusion protein forms of IL-12 prolonged survival and led to complete tumor regression.