AbstractWe describe a rapid automated technique for colorimetric in situ DNA hybridization using the Code-On Immuno-DNA Slide Stainer. We report recent advances in positively charged glass slide technology, rapid limolene/xylene dewaxing, stable ribonuclease and pepsin predigestion, heat cycling during hybridization, and alkaline phosphatase enhancement. These amplify the signal to noise ratio of colorimetric molecular hybridization to the point where complete one hour assays can be performed from dewaxing to counterstaining by automation. Both biotin labeled oligonucleotide and nick translated DNA probes can localize their viral targets in situ with this single, mechanized protocol at the rate of one slide per minute. (The J Histotechnol14:219, 1991)