Adipose tissue synthesizes factors involved in the body's homeostasis. Thus, measurements of messenger ribonucleic acid (mRNA) concentrations are important to study the involvement of adipose tissue in various physiological and pathophysiological conditions, in particular in obesity. Because adipose tissue is highly heterogeneous, containing both a stromal and an adipocyte compartment, each one having different cellular composition and functional capacities, in situ hybridization is a powerful tool to analyze the discrete expression of the mRNAs coding for the various factors synthesized within this tissue. Presented here is a detailed protocol for in situ hybridization of mRNAs in adipose tissue using ³⁵S-labeled single-stranded probes with sufficient details for the readers unfamiliar with histologic techniques. Included are details of tissue sectioning and preparation, probe synthesis, hybridization reaction, and macro- and microscopic signal detection.