We report the establishment of human-human hybridomas producing monoclonal antibody of predefined antigenic specificity. The U-266 human myeloma cell line was incubated in the presence of 8-azaguanine, and a rapidly growing, 8-azaguanine-resistant, hypoxanthine/amethopterin/thymidine (HAT) medium-sensitive mutant line, U-266AR1, was selected. These cells were fused with lymphoid cells from uninvolved spleens removed at staging laparotomy from patients with untreated Hodgkin's disease who had been previously sensitized to the chemical allergen 2,4-dinitrochlorobenzne. Hybrid cell cultures growing in HAT medium were screened for IgG production. Positive cultures were selected and their supernatants were tested in a solid-phase radioimmunoassay for reactivity with dinitrophenyl hapten coupled to bovine serum albumin. Cultures producing specific antibody were subcloned and expanded, and their antibody products were shown to be monoclonal by biosynthetic labeling and sodium dodecyl sulfate/polyacrylamide gel electrophoresis.