A new EBV positive human B-cell line, BONNA-12 was established from splenic cells of a patient with a hairy cell leukemia (HCL). BONNA-12 cells grew spontaneously and formed colonies in semisolid media. Although the BONNA-12 cell line was identical with the patient's spleen cells by HLA analysis and Southern blot examination of minisatellite DNA patterns, the immunoglobulin heavy and light chain rearrangement patterns differed from the original HCL. Cytogenetic analysis of the BONNA-12 cell line demonstrated in the major cell clone a 47, X, -Y, +9, +12 karyotype. Trisomy 12 is a characteristic abnormality in chronic lymphocytic leukemia that also rarely occurs in HCL. The BONNA-12 cell line is of potential value in the study of trisomy 12 in chronic B cell malignancies.