Summary We have translated the HLA-B27 heavy chain in vitro and studied its assembly with β2-microglobulin and peptide in microsomes from human cells. The assembly process requires ATP. However, the translocation of peptide across the endoplasmic reticulum (ER) membrane does not require ATP, and binding of biotinylated peptide to BiP, and ER luminal protein, occurs after ATP depletion. Proteinase K treatment of the microsomes does not block peptide translocation. Thus, ATP is required in the lumen of the ER for efficient assembly to occur. Microsomes prepared from Raji and T1 cells show similar levels of assembly, whereas assembly of T2 microsomes in 10-fold lower. This difference remains after peptide stimulation of assembly. The inefficient assembly in T2 microsomes is not due to impaired peptide translocaton across the ER membrane, as no difference was found compared with micosomes from T1 cells. Instead, the defect seems to reside in the lumen of the ER.