C2-alpha-Mannosyltryptophan was discovered in RNase 2 from human urine, representing a novel way of attaching carbohydrate to a protein. Here, we have addressed two questions related to the biosynthesis of this modification: (i) is C-mannosylation part of the normal intracellular biosynthetic route, and (ii) how general is it, i.e. which organisms perform this kind of glycosylation? To answer the first question, RNase 2, which is identical to the eosinophil-derived neurotoxin, was isolated from intracellular stores of cultured human HL-60 cells. The enzyme was C-mannosylated at Trp-7, showing that the modification occurs intracellularly, before secretion of the protein. The second question was investigated by immunological and chemical analysis of RNase 2 purified from the supernatant of transiently transformed cells from different organisms. This revealed that C-mannosylation occurs in cells from man, green monkey, pig, mouse, and hamster. The observation that pig kidney cells contain the machinery for C-mannosylation of Trp-7 of human RNase 2 but that the homologous RNase from porcine kidney is not a substrate, since it does not contain a tryptophan at position 7, strongly suggests that C-mannosylated proteins other than RNase 2 exist. Recombinant RNase 2 isolated from insect cells, plant protoplasts, and Escherichia coli was not C-mannosylated. These results not only form the basis for further studies on the biochemical aspects of C-mannosylation but also have implications for the choice of cells for production of recombinant glycoproteins.