We applied a new strategy for the detection of N-ras gene mutations based on LightCycler technology. We designed two sets of amplimers and internal hybridization probes representing N-ras codons 12/13 and codon 61, respectively. Genomic DNAs from 134 childhood acute lymphoblastic leukemia (ALL) patients (83 common ALL, nine pre-pre-B ALL, 19 pre-B ALL, 23 T-ALL) were amplified, followed by the analysis of the melting temperatures of the PCR products on the LightCycler. PCR products exhibiting an abnormal melting characteristic were directly sequenced. Sequence analyses unravelled nucleotide substitutions at codon 12 in 10 patients, at codon 13 in three, and at codon 61 in one case. The incidence of N-rasmutations (10%) is compatible with previous reports. The LightCycler technology facilitates the rapid analysis of other genes exhibiting hot spot mutations in human malignancies.