Tissue microarrays enable the rapid histological localization of gene expression in hundreds of archival samples by in situ hybridization. However, the scoring of tissue microarray data may be influenced by intra- and inter-observer variations, and categorizing continuous variables risks discarding potentially meaningful information. Quantitation imposes a greater degree of objectivity, is more reproducible than subjective discriminations, and facilitates the communication and clarity of definitions. Phosphorimaging has been successfully used to quantitate the hybridization signal intensity from arrayed tissues. The process is rapid and has a wide dynamic range, surpassing the densitometric analysis of autoradiograms. This paper presents a detailed method for quantitative isotopic in situ hybridization on formalin-fixed paraffin-embedded tissue microarrays. In addition, the method includes a protocol for the development of synthetic agarose cores to control for the specificity and sensitivity of hybridization.