In this article we have described our protocol for in situ cDNA:mRNA hybridization and a variety of methodological considerations we have entertained to optimize the procedure. It should be stressed that each different system will have its own special characteristics and require optimization to obtain maximal and reproducible signals. We believe that by following a methodological logic as outlined here, researchers should be able to establish the in situ cDNA:mRNA hybridization protocol with their probes and tissue systems.