Multiple color spectral karyotyping (SKY) has been proven to be a very useful tool for characterization of the complex rearrangements in cancer cells and the de novo constitutional structural abnormalities. The sensitivity of SKY in detecting interchromosomal alterations was assessed with 10 constitutional translocations involving subtelomeric regions. Among the 13 small segments tested, 9 were clearly visualized and 8 were unambiguously identified by SKY. Fluorescence in situ hybridizations (FISH) with subtelomeric probes confirmed the reciprocity in three of the four translocations in which a small segment was not detectable by SKY. On the basis of resolution level of G-banding and the information obtained from the FISH analysis, the minimum alteration that SKY can detect is estimated to be 1,000-2,000 kbp in size with the currently available probes. This study has demonstrated the power, but also the limitations, of SKY in detecting small interchromosomal alterations, particularly those in subtelomeric regions.