Interleukin-12 (IL-12) is a heterodimeric cytokine that consists of two structurally unrelated subunits, P35 and P40. However, when expressed alone in Chinese hamster ovary (CHO) cells. murine P40 showed two species of different molecular weights under nonreducing conditions, a monomeric form of (45 \mathrm{kDa}) and a homodimer of (>97 \mathrm{kDa}). Under reducing conditions the two forms migrated as an identical array of species of (40-45 \mathrm{kDa}). The monomer was separated from the homodimer under nonreducing conditions by heparin affinity chromatography and the disulfide bond structures of both species were determined by peptide mapping. Edman sequencing, and mass spectrometry. The peptide maps of the two species were identical except for a single peak that changed retention time. Sequer cing showed that this peak contained two peptides of identical sequences in both maps. Miss spectrometric analysis of the peak from the (>97-\mathrm{kDa}) species revealed an ion of double th e expected mass, thus indicating that the peptide pair had dimerized. Mass analysis of the peak from the 40)to (45-\mathrm{kDa}) species showed that the peptide pair contained a mass difference t at corresponded to that of an extra cysteine and which disappeared upon reduction. Amino acid analysis confirmed that the monomeric form of rmP4() is modified by a reducible cysteine. Structural analysis of the remainder of the cysteine-containing peaks showed that both species of rmP40 contained the same set of intramolecular disulfide bonds. The murine P40 romodimer arises from formation of a single intermolecular disulfide bond at (\mathrm{Cys}^{175}). In the monomeric P40), however, this cysteine is capped by an additional cysteinc. Purified rmP40 monomer and homodimer inhibited the IL-12-dependent induction of interferon- (\gamma). but neither appeared capable of inducing IL-12-like biological activity. o1995 Academic Press. Inc.