We previously observed that in a mutant B lymphoblastoid cell line which has a homozygous HLA-DR alpha deletion, DR beta-chains appeared to be unstable. In the present study, we have studied the pathway that leads to degradation of unassembled DR beta-chains. Unassembled DR beta-chains are degraded rapidly in the DR alpha deletion mutant cells, compared with the assembled DR heterodimers present in non-mutant cells. Accelerated DR beta turnover in 9.22.3 cells is specific; class I molecules in these DR alpha-deficient cells turned over slowly. DR beta-chains assemble with Ii in the DR alpha deficient cell line, but this did not protect DR beta-chains from degradation. The maturation of unassembled beta-chains is arrested before their reaching the medial Golgi compartment, and this degradation proceeds by a nonlysosomal, nonendosomal pathway. Degradation of DR beta-chains is blocked when cells are cultured at 16 degrees C, a temperature known to prevent vesicular transport between the endoplasmic reticulum (ER) and the Golgi apparatus. Degradation is also inhibited by carbonyl cyanide m-chlorophenylhydrazone, a drug that is also known to inhibit protein transport from the ER. The results, taken together, suggest that degradation of unassembled DR beta-chains occurs by a nonlysosomal, nonendosomal pathway which involves transport of DR beta-chains out of the ER.