In situ hybridization is a powerful technique for examining changes in mRNA levels. Its primary advantage over the Northern blot and reverse transcription polymerase chain reaction is the ability to localize specific species of mRNA to a particular cell population in a heterocellular system. This consideration is important when studying gene expression, especially in areas of high cellular heterogeneity, such as the central nervous system, where the differentiation between neuronal and glial gene expression is critical in evaluating the effects of physiological or pathological stimuli. We have used radioactive in situ hybridization to study changes of mRNA levels in several motoneuron populations following axonal injury. Considering that many neuronal populations have multiple targets of innervation, we used in situ hybridization in a manner that focused on the examination of changes in gene expression at the single cell level. This chapter describes the in situ hybridization protocol that our laboratory has used to demonstrate alterations in gene expression in specific motoneurons whose cell bodies are localized to the central nervous system.