Improved techniques for detecting in situ hybridization signals on fluorescent G-bands are described. Prior to hybridization of biotinylated DNA probes, chromosomes with replication G-bands were treated with formaldehyde and denatured with an alkaline solution. These steps preserve chromosome morphology well, and enable us to simultaneously demonstrate fluorescein hybridization signals and G-banded chromosomes stained with propidium iodide. This method is useful for precise chromosomal localization of DNA markers.