This protocol describes pulsed-field gel electrophoresis (PFGE), a method developed for separation of large DNA molecules. Whereas standard DNA gel electrophoresis commonly resolves fragments up to approximately 50 kb in size, PFGE fractionates DNA molecules up to 10 Mb. The mechanism driving these separations exploits the fact that very large DNA molecules unravel and "snake" through a gel matrix, and such electrophoretic trajectories are perturbed in a size-dependent manner by carefully oriented electrical pulses. PFGE has enabled the rapid genomic analysis of microbes and mammalian cells, and motivated development of large-insert cloning systems such as bacterial and yeast artificial chromosomes. As such, this protocol includes descriptions of two types of PFGE instrumentation (not commercially available), along with detailed instructions for their operation. Additionally, this protocol provides basic instructions for the preparation of intact chromosomal DNA from several types of organisms. PFGE takes 2-3 days, excluding sample preparation.