We examined chromosome 3 in 32 childhood acute lymphoblastic leukemia (ALL) bone marrow samples. Using interphase multipoint FISH (mp-FISH), which was developed by our group, with 42 chromosome 3-specific probes, we detected clonal chromosome 3 aberrations in 4 T-cell ALL (T-ALL) cases. Four out of seven T-ALL cases carried 3q trisomies. One T-ALL case carried either trisomy 3 (in 15% of the cells) or a 23-megabase (Mb) 3p13 approximately p12 deletion in a different subpopulation of cells of 32%. Another T-ALL case had either 3q trisomy in 11% or a 12-Mb 3p12 approximately p13 deletion in 19% of the cells. The deletions were overlapping. In both cases, the majority of the bone marrow cells (47 and 70%, respectively) were normal chromosome 3 disomics. The interstitial deletions detected harbor a known homozygous deletion region between 72.6 and 78.8 Mb, which has been described in lung and breast tumors and contains the DUTT1/ROBO1 tumor suppressor gene. These deletions detected by mp-FISH would have remained unnoticed by conventional cytogenetics and multiplex FISH, as well as by current methods based on total tumor DNA analysis such as comparative genomic hybridization (CGH), array CGH, and loss of heterozygosity (LOH).