In situ hybridization allows the detection of specific gene transcripts in tissues, cells or, chromosomes. In the cardiovascular field, this powerful and rapid methodology provides precious insights into the complex gene organization and expression within an heterogeneous cell population. This technique is particularly useful to elucidate the genes and pathways involved in cardiac cells processes (differentiation, proliferation, apoptosis) or in the development of cardiovascular pathologies. In situ hybridization allows the precise localization of gene transcripts to the different heart regions and to individual cell types such as working cardiomyocytes, cells from conductive tissues and blood vessels displaying specific functions. This chapter describes the different technical procedures that are of crucial importance to carry on sensitive and specific in situ hybridization experiments in heart samples. The detection of transcripts within paraformaldehyde-fixed, paraffin-embedded cardiac tissue samples is illustrated here with the detection of cardiac sphingosine-1-phosphate receptor expression.