In general, an elevated expression of beta 3-galactosyltransferase (beta 3GalT) activity contributed by beta 3GalT5 correlates well with increased biosynthesis and expression of type 1 chain (Gal beta 1-3GlcNAc beta 1-) derivatives such as Lewis A and sialyl Lewis A, which are mostly recognized as terminal epitopes and not further extended. Most known beta 3-N-acetylglucosaminyltransferases show a higher activity toward extending type 2 chain (Gal beta 1-4GlcNAc beta 1-), and an over-expression of beta 3GalT5 could suppress the formation of the type 2 chain poly-N-acetyllactosaminoglycans. The potential of extending instead the predominant type 1 chain termini synthesized under such circumstances was, however, not investigated, partly due to technical difficulty in unambiguous identification of extended type 1 chains. Using an advanced mass spectrometry-based glycomic mapping and glycan sequencing approach, we show here that type 1 chains carried on the lacto-series glycosphingolipids of colonic carcinoma cells can be extended when the endogenous beta 3GalT activity relative to competing beta 4GalT activity, as defined against a common GlcNAc beta 1-3Gal beta 1-4Glc acceptor, is sufficiently high, as found in Colo205 and SW1116, but not in DLD-1 cells. In support of this positive correlation, the lacto-series glycosphingolipids isolated from stably transfected DLD-1 clones over-expressing beta 3GalT5 were shown to comprise fucosylated dimeric type 1 chains, whereas a mock transfectant and the DLD-1 parent carried only fucosylated dimeric type 2 chains on their lactosylceramides. It suggests that while the natural expression of extended type 1 chain is likely to be determined by many contributing factors including the relative amounts of competing glycosyltransferases and the UDP-Gal level, the enhanced expression of beta 3GalT5 is sufficient to promote in vivo extension of type 1 chains by furnishing a significantly higher amount of type 1 chain precursors relative to competing type 2 chains.