This paper describes the in vitro production of the human alloantibody 454A.C5 with HLA-DQw2 specificity produced by a stable EBV-transformed cell line. Immune B cells for transformation were generated by planned immunization of a blood donor volunteer with repeated HLA-immunizations over the course of 9 yr. Reactivity of 454A.C5 was in exact concordance with the presence of the DQw2 antigen as defined by microcytotoxicity assay on a panel of 21 B lymphoblastoid cell lines with well-characterized HLA antigens. Family studies showed the segregation according to the HLA-DQw2 haplotype; thus we demonstrate by serological and formal genetics criteria, that the alloantibody is specifically directed against the DQw2. 454A.C5 antibody may be employed as a monospecific HLA typing reagent.