Over the years, in situ hybridization has been used for visualizing spatial gene expression patterns. Direct comparison of the expression patterns of different genes is useful for studying various biological activities in many situations. If we can distinguish signals derived from different probes in the same hybridization reaction, the localization of multiple gene transcripts can be detected simultaneously. In this chapter, we describe a two-color in situ hybridization procedure, which enables us to compare the expression patterns of two genes in a single tissue section. First, we explain how to prepare RNA probes and tissue samples. Then, we explain how to perform an in situ experiment, including pre-hybridization, hybridization, post-hybridization, and detection steps. A stepwise detection procedure is used to obtain the two color signals.