Leukemia inhibitory factor (LIF) is a pluripotent growth factor which acts in various cell systems. LIF is a glycoprotein containing six putative N-glycosylation sites. We established Chinese hamster ovary (CHO) cell lines to evaluate the biological roles of the N-glycosylation in rat LIF (rLIF). The bioactivity of rLIF was evaluated in two different bioassay systems using F9 and DA-1a cells. Employing site directed mutagenesis, six N-glycosylation-deficient LIF mutants were generated by replacing each asparagine residue (N) (at positions N9, N34, N63, N73, N96, and N116) by glutamine (Q). The resultant mutants showed similar activity in the bioassay using F9 cells. However, N34Q was about 3 times more potent than the wild-type rLIF in the assay using DA-1a cells. These findings suggest that the presence of N-glycan at N34 suppresses cell proliferation. In contrast, N63Q was about 2.5 times less potent than the wild-type rLIF indicating the pivotal role of N63 glycosylation for rLIF bioactivity. Taken together, our data suggest that the N-glycans of LIF play different roles depending on the cell line and that glycosylation of each specific residue contributes differently to its bioactivity.