A new cell line (NALL-1) was established in vitro from the peripheral blood of a patient with acute lymphoblastic leukemia (ALL). NALL-1 cells had neither properties of T and B cells nor Epstein-Barr virus (EBV). Many characteristics of the NALL-1 line were distinct from those of numerous EBV-positive lymphoblastoid cell lines previously established. The NALL-1 line grew as single-cell suspension cultures with a doubling time of 72 hours and consisted of primitive lymphoid cells with large nuclei, prominent nucleoli and scanty cytoplasm. Chromosome analysis of NALL-1 cells revealed a hypodiploid karyotype with a minute marker chromosome. NALL-1 cells are considered to have originated from the donor's leukemic cells on the basis of their cytoplasmic, morphologic and functional features.