Gene regulation in higher eukaryotes frequently involves physical interactions between genomic sequence elements tens of kilobases apart on the same chromosome but can also entail interactions between different chromosomes. Chromosome Conformation Capture (3C) is a powerful tool to identify such interactions. 3C technology is based on formaldehyde crosslinking of chromatin, followed by restriction digestion and intramolecular ligation. Quantitative detection of ligation products by PCR (qPCR; not discussed in this protocol) provides insight into the interaction frequencies between chromosomal fragments and thereby the spatial organization of a genomic region. Detailed 3C protocols have been published for yeast and mammals. However, these protocols cannot simply be transferred to plant tissues. In this paper, we provide a maize-specific 3C protocol and present a general strategy to systematically optimize the protocol for other plants. Once the technique and appropriate controls are established, the 3C procedure (including qPCR) can be completed in 5-7 d.