Y-box or inverted CCAAT box-binding proteins are multifunctional regulators of transcription and translation of several genes. Although YB-1 has been shown to play a key role in cell cycle, to date, there is no direct evidence. We disrupted one allele of Chk-YB-1b in a chicken pre-B lymphocyte cell line, DT40. Compared to wild-type DT40 cells, these heterozygous DT40YB1b(+/-) cells with one copy of the wild-type Chk-YB-1b allele showed multiple abnormalities, which include slower rate of growth, abnormal cell morphology, increased cell size, and increased genomic DNA content. These phenotypic defects resemble those cells that have a block in G2 and/or mitosis (G2/M). In addition, we have observed that a fraction of these heterozygous DT40YB1b(+/-) cells undergo apoptosis. In conclusion, we have discovered major defects in the G2/M phase of cell cycle in YB-1 knocked-out heterozygous mutant cells, providing for the first time direct evidence establishing a crucial role for YB-1 in cell proliferation. (C) 2002 Elsevier Science (USA). All rights reserved.