Deaminoneuraminic acid (KDN, 2-keto-3-deoxy-d-glycero-d-galacto-nononic acid) is a member of the family of sialic acids in which an acylamino group at the C-5 position of N-acylneuraminic acid (Neu5Acyl) is replaced by a hydroxyl group. It has recently been shown that KDN is synthesized de novo from its precursor, mannose (Man), in trout testis (Angata, T., Nakata, D., Matsuda, T., Kitajima, K., and Troy, F. A. (1999) J. Biol. Chem. 274, in press). In this study, we examined the effect of extracellular free Man on biosynthesis of KDN in mouse melanoma B16 and African green monkey kidney COS-7 cell lines. The following new findings are reported. First, the levels of free and bound forms of KDN increased when the cells were cultured in the presence of 20 mM Man. The level of intracellular free KDN in COS-7 and B16 cells increased 47- and 66-fold respectively, compared with the levels in control cells. Second, the elevated expression of free KDN was proportional to the intracellular concentration of free Man. Third, KDN 9-phosphate (KDN-9-P) synthase, which condenses Man 6-phosphate and phosphoenolpyruvate (PEP), forming KDN-9-P, was detected in cell lysates from both cell lines. Fourth, the de novo synthesis of KDN in both cell lines in the Man-rich media was unaffected by the addition of N-acetylmannosamine (ManNAc), the hexosamine precursor for synthesis of N-acetylneuraminic acid (Neu5Ac). These results show that KDN is synthesized using free Man as its hexose precursor in these mammalian cells. Thus, the KDN biosynthetic pathway utilizes enzymes distinct, at least in part, from those involved in Neu5Ac biosynthesis. This is the first report showing that in vivo synthesis of KDN can be manipulated by growing cells in the presence of Man. This now provides a useful method to study the metabolism and function of the KDN glycotope.