We have identified a novel pre-TCR isoform that is structurally distinct from conventional pre-TCR complexes and whose TCR beta chains are inaccessible to anti-TCR beta antibodies. We term this pre-TCR isoform the MB (masked beta)-pre-TCR. Pre-T alpha (pT alpha) subunits of MB-pre-TCR complexes have a larger apparent mol. wt due to extensive modification with O:-linked carbohydrates; however, preventing addition of O-glycans does not restore antibody recognition of the TCR beta subunits of MB-pre-TCR complexes. Importantly, accessibility of TCR beta chains in MB-pre-TCR complexes is restored by filling in the 'missing' variable (V) domain of pT alpha with a V domain from TCR alpha. Moreover, the proportion of pre-TCR complexes in which the TCR beta subunits are accessible to anti-TCR beta antibody varies with the cellular context, suggesting that TCR beta accessibility is controlled by a trans-acting factor. The way in which this factor might control TCR beta accessibility as well as the physiologic relevance of TCR beta masking for pre-TCR function are discussed.