We have examined the requirement for beta 2-microglobulin (beta 2m) in the intracellular transport of murine class I histocompatibility molecules to the cell surface. R1E cells that are defective in the synthesis of beta 2m were transfected with either the class I H-2Kb or H-2Db genes alone, or together with the beta 2m gene. Kb or Db heavy chains synthesized in the presence of beta 2m were transported rapidly through the cell and expressed efficiently at the cell surface. In the absence of beta 2m, no "free" Kb heavy chains were detectable at the cell surface and their intracellular transport was blocked at an early stage. In contrast, a significant quantity of "free" Db heavy chains could be detected at the cell surface as we have reported previously. However, we have shown here that defects in intracellular transport were apparent in that the majority (approximately 70%) of newly synthesized Db heavy chains accumulated intracellularly and were degraded. Therefore, although Kb and Db heavy chains differ in their abilities to be expressed at the cell surface in the absence of beta 2m they both require association with beta 2m for efficient intracellular transport. In addition, R1E cells transfected with a deletion construct of the Kb gene expressed a truncated molecule lacking the alpha 3 extracellular domain (Kb-3) at the cell surface, but, like free Db, most newly synthesized Kb-3 molecules accumulated intracellularly. The free Kb, Kb-3, and Db heavy chains were not recognized by most mAb specific for Kb and Db, respectively. Therefore, even the transported forms of free Db and Kb-3 were not native in conformation, which is surprising given the current view that correct folding is essential for intracellular transport. Interestingly, the free Db and Kb-3 heavy chains that reached the cell surface differed in their detergent binding properties from those retained within the cell. This suggests that the transported heavy chains may have folded differently thus allowing their export to the cell surface.