A number of important nuclear processes including replication, recombination, repair, and transcription involve the interaction of soluble nuclear proteins with DNA assembled as chromatin. Recent progress in a number of experimental systems has focused attention on the influence chromatin structure may exert on gene regulation in eukaryotes. With the advent of new technologies for the analysis of chromatin structure in vivo, studies evaluating the influence of chromatin structure on gene transcription have become feasible for a number of systems. This article serves as an introduction to the use of restriction endonucleases to define nucleosomal organization and characterize changes in this organization that accompany transcriptional activation in vivo. The procedure includes the isolation of intact transcriptionally competent nuclei, limited digestion with specific restriction endonucleases, and purification of the DNA. This DNA serves as the substrate for a linear amplification using single primers that generate enzyme-specific DNA fragments, which are then resolved by electrophoresis. Specific examples related to our studies of the influence of chromatin structure on steroid hormone regulation of transcription from the mouse mammary tumor virus promoter are provided to illustrate this technique and several novel variations. Alternative methods for analysis of chromatin architecture using DNase I, micrococcal nuclease, permanganate, and methidiumpropyl-EDTA-iron(II) are also described. Through the use of these methodologies one is able to determine both the translational and the rotational positions for a given nucleosome as well as quantify changes at a specific nucleosome in response to regulatory and developmental signals.