Immunofluorescence (IF) and Fluorescence in situ Hybridization (FISH) are conventional methods used to study the structure and organization of metaphase chromosomes and interphase nuclei. Using these techniques, the locations of whole chromosome territories, chromatin subdomains, and specific DNA sequences can be evaluated at kilobase or megabase resolution. Even higher resolution of the spatial relationships of proteins and DNA can be achieved using combined IF-FISH on extended chromatin fibers. This method of optical mapping is a powerful system for localizing molecular probes along released chromatin fibers and visualizing small (<20 kb) or large (20-5,000 kb) chromosomal domains. Chromatin fiber analysis can fill the gaps in resolution between classical chromosome studies and molecular analyses, such as chromatin immunoprecipitation (ChIP) that evaluates chromatin organization at the level of single or multiple nucleosomes. In this chapter, the conceptual and technical aspects of chromatin fiber IF-FISH are presented, along with examples of successful applications.