To simultaneously detect gene expression for bombesin/gastrin-releasing peptide (GRP) and its receptor in routinely processed tissue sections, we developed a protocol for non-radioactive double in situ hybridization (ISH) method.We used digoxigenin-labeled cRNA probe for GRP and a biotinylated cRNA probe for GRP-receptor (GRP-R) on frozen and/or formalin-fixed, paraffin-embedded sections of rat brain. The probes were applied singly and mixed (double ISH) and the signals visualized with two distinct chromogens. Antidigoxigenin-alkaline-phosphatase conjugate and nitro blue tetrazolium, producing a blue color reaction, was used to visualize GRP mRNA, and for GRP-R mRNA, avidin-alkaline phosphatase and vector red, were used resulting in a vivid red color. To unmask the signals on formalin-fixed, paraffin-embedded tissue, the sections were pretreated with proteinase VIII.Strong specific signals for GRP and/or GRP-R were detected in rat-brain neurons. The localization and intensity of the signal was comparable on frozen and routinely processed sections, although the latter showed better cytology and resolution. The overall distribution of GRP and GRP-R mRNAs in rat brain was similar to that previously reported with single ISH using radioactive-labeled probes. Double ISH revealed colocalization of the two mRNAs in some neurons with the expression of GRP mRNA often coexpressed with GRP-R mRNA. However, the expression of GRP-R mRNA did not always correlate with GRP mRNA signal.The nonradioactive, double ISH method is a relatively simple and sensitive procedure applicable to routinely processed tissues. This method may be suitable for studies on temporal and spacial distribution of peptide(s) and peptide receptor gene expression in health and disease.