The t(2;5) (p23;q35) that is frequently detected in anaplastic large cell lymphoma (ALCL) fuses the nucleophosmin (NPM) gene on chromosome 5 to a novel tyrosine kinase gene designated anaplastic lymphoma kinase (ALK) on chromosome 2. The fusion of NPM and ALK genes results in the production of chimeric transcripts containing NPM amino-terminal sequences fused to the ALK carboxy-terminal catalytic domain. Because fusion transcripts and proteins in almost all t(2;5)-positive cell lines and tumors are identical, it is likely that the chromosomal breaks involve the same introns of NPM and ALK genes. We have previously developed a long-range genomic DNA-PCR assay to amplify the genomic NPM-ALK break points. Using high-molecular-weight DNA extracted from 2 ALCL cell lines and from 9 primary ALCLs known to be t(2;5)-positive, we have demonstrated that all 11 amplicons were of different sizes, suggesting that the t(2;5) break points were unique and involved the same introns on both chromosomes. We decided to confirm this and map the t(2;5) break points by genomic DNA sequencing. Using the same long-range DNA-PCR technique, primers from the ALK locus, and normal genomic DNA, we sequenced the ALK intron involved in t(2;5). We subsequently sequenced all 11 amplicons from t(2;5)-positive ALCL cell lines and tumors. Comparison of the sequences derived from ALCL amplicons with the published sequences of intron 4 from the NPM locus (910 bp) and with the newly sequenced intron from the ALK locus (1935 bp) accurately mapped all break points and demonstrated that their nucleotide sequences were unique. We conclude that the genomic t(2;5) break points can be easily mapped by sequencing the amplicons generated from genomic DNA with long-range PCR and that they are unique for each patient. The sequences of the break points and of the newly identified ALK intron may be useful in the construction of patient-specific primers for monitoring and determination of the clinical relevance of minimal residual disease.