We developed a new HNPP-azo dye method for detection of fluorescence in situ hybridization (FISH) signals on Q-banded chromosomes by use of a newly synthesized fluorochrome, HNPP (3-hydroxy-N-2'-biphenyl-2-naphthalenecarboxamide phosphate ester), which reacts enzymatically with alkaline phosphatase and azo dye. The FISH staining method permits simultaneous detection of orange HNPP signals on chromosomal sites labeled by Q-banding, allowing the assignment of small (440-1,200 bp) probes.