The beta-chain of human interleukin 12 (IL-12) contains at position 319-322, the sequence Trp-x-x-Trp. In human RNase 2 this is the recognition motif for a new, recently discovered posttranslational modification, i.e., the C-glycosidic attachment of a mannosyl residue to the side chain of tryptophan. Analysis of C-terminal peptides of recombinant IL-12 (rHuIL-12) by mass spectrometry and NMR spectroscopy revealed that Trp-319beta is (partially) C-mannosylated. This finding was extended by in vitro mannosylation experiments, using a synthetic peptide derived from the same region of the protein as an acceptor. Furthermore, human B-lymphoblastoid cells, which secrete IL-12, were found to contain an enzyme that carries out the C-mannosylation reaction. This shows that nonrecombinant IL-12 is potentially C-mannosylated as well. This is only the second report on a C-mannosylated protein. However, the occurrence of the C-mannosyltransferase activity in a variety of cells and tissues, and the presence of the recognition motif in many proteins indicate that more C-mannosylated proteins may be found.