Translocations between two specific chromosomes are important markers for many human malignancies. Previously, the detection of translocations involving random breakpoints between two specific chromosomes could only be accomplished by banding techniques, which are severely labor intensive and require highly trained technicians. The three-color chromosome painting approach described in this paper was developed in our laboratory to detect translocations between two specific human chromosomes rapidly and accurately, while simultaneously revealing the nonhybridized chromosomes. Because this method efficiently detects translocations involving breakpoints anywhere on the targeted chromosomes, it is ideal as a screening tool for chromosome-specific translocations.