To determine whether genetic material in 5q13.3 breakpoint region changed in the course of establishing the cell line with a constitutional chromosomal inversion inv(5)(p13.1q13.3) associated with hairy cell leukemia, double color fluorescence in situ hybridization (FISH) on metaphase, interphase and DNA fibers were performed in cells of the cell line with the cosmids cCI5-216 and cCI5-267 DNA probes labeled by either biotinylate or digoxigenin. It showed that the cell line gave the same results as those of the original cells, which indicated that no change of the genetic material at 5q13.3 breakpoint region occurred. The cell line is valuable to reveal the molecular pathogenesis of hairy cell leukemia.