Acute promyelocytic leukemia (APL) is characterized cytogenetically by a balanced reciprocal chromosomal translocation t(15;17) (q22;q21). This translocation involves the retinoic acid receptor-alpha (RAR-alpha) on chromosome 17 and the promyelocytic leukemia locus (PML) on chromosome 15 and results in the transcription of novel fusion messenger RNAs. In this study, pulsed-field gel electrophoresis (PFGE) was applied to the detection of the t(15;17) translocation in twenty-six clinical specimens cytologically diagnosed by French-American-British criteria as APL. This technique could readily be applied to both fresh and nonviably frozen tumor samples. In 24 of 26 samples, rearrangements of the PML and RAR-alpha, loci could be detected by Southern blotting after digestion with MluI and BssHII. Furthermore, co-migration of the rearranged fragments, detected by hybridization to probes for the PML and RAR-alpha genes, demonstrated that these loci were juxtaposed. The translocation was detected in specimens at the time of initial diagnosis, on differentiation therapy with retinoic acid and at the time of relapse. The diagnostic accuracy was compared to cytogenetics and the reverse transcriptase-polymerase chain reaction for the novel PML-RAR-alpha fusion transcript. The samples from two patients were negative by all three diagnostic methods, and both of these patients failed to respond to all-trans retinoic acid. In the other 24 APL samples, cytogenetics was positive in only 76.9% of the cases, whereas both reverse transcriptase-polymerase chain reaction and PFGE methods detected the translocation in 100% of the cases. Thus, PFGE can readily detect the t(15;17) translocation in both viable and nonviable clinical specimens and can improve the diagnostic accuracy of morphology and cytogenetics in APL. In contrast to conventional electrophoresis based on rearrangement of RAR-alpha, the ability to demonstrate directly co-migration of the PML and RAR-alpha loci enables this method to distinguish the t(15;17) translocation from variant translocations such as the t(11;15). Because PFGE can be performed on nonviable, frozen tumor samples, it could be diagnostically useful in APL when the RNA-based reverse transcriptase-polymerase chain reaction cannot be performed.