Glycosylation is known to be one of the most prevalent co- and post-translational modification of proteins, affecting the physicochemical/physiological properties of cognate proteins. It is also known that glycans can also occur as a “free”, unconjugated form. Recent studies have clarified that there are multiple different pathways involved in the formation of free oligosaccharides (1). For instance, intracellular free N-glycans, free oligosaccharides structurally related to asparagine-linked (N-linked) glycans, are mainly formed by hydrolysis of oligosaccharides in mammalian cells (2, 3), while they are predominatly formed by the action of the cytoplasmic peptide:N-glycanase in budding yeast, S. cerevisiae (4). There are also “extracellular” free oligosaccharides found in various animal sera (5). Their formation mechanism should be distinct from that for intracellular glycans. In this talk, I will summarize the current state of our knowledge concerning the formation of free oligosaccharides in mammalian cells and yeast.