The rates of graft-versus-host disease (GVHD) and rejection are significantly higher among recipients of unrelated donor marrow (BM) than in recipients of marrow from HLA-identical siblings, even when donors and recipients are mixed lymphocyte culture (MLC) compatible and serologically and Dw identical. It has been hypothesized that phenotypically silent HLA class I and DP sequence mismatches might be associated with these differences, but little is known about their incidence. We have sequenced the HLA-A, HLA-B, HLA-C, HLA-DPA1, and HLA- DPB1 genes expressed by 12 unrelated marrow transplant pairs, 11 of whom were molecularly matched at DRB, DQA1, and DQB1 loci. Nine of these pairs were also HLA-A and HLA-B matched by serology. Six of these nine “HLA-identical” pairs were HLA-A (2 of 6), HLA-B (1 of 6), and HLA- C (6 of 6) mismatched at the sequence level. The mismatched class I alleles of all these pairs had strikingly different sequence motifs in the six specificity pockets of their antigen recognition site, and in five pairs they also had sequence differences at positions implicated in T-cell receptor (TCR) binding. Two of the three pairs who were serologically mismatched for one HLA-A or HLA-B antigen were also sequence mismatched at HLA-C. Finally, 10 of 11 pairs tested expressed different DP sequences. These data indicate that HLA class I, especially HLA-C, and DP sequence mismatches are frequent among unrelated subjects defined as HLA identical by current typing methods. We speculate that these sequence differences may explain, at least in part, the higher incidence of acute GVHD and rejection in unrelated BM transplantation as opposed to transplantation between HLA-identical siblings. Because of their high frequency, the role of HLA-A, HLA-B, HLA-C, and HLA-DP mismatches in transplantation outcome is now amenable to direct study.