The organization of the mitochondrial genome in plants is not well understood. In maize mitochondrial DNA (mtDNA) several subgenomic circular molecules as well as an abundant fraction of linear molecules have been seen by electron microscopy. It has been hypothesized that the circular molecules are the genetic entities of the mitochondrial genome while the linear molecules correspond to randomly sheared mtDNA. A model has been proposed that explains the mechanism of generation of subgenomic circles (of a predictable size) by homologous recombination between pairs of large direct repeats found on a large (approximately 570 kb for the fertile (N) cytoplasm) master circle. So far the physical entities of the mitochondrial genome, as they exist in vivo, and the genes they carry, have not been identified. For this purpose, we used two gel systems (pulsed field gel electrophoresis and Eckhardt gels) designed to resolve large DNA. Large DNA was prepared from the Black Mexican Sweet (BMS) cultivar. We resolved several size classes of mtDNA circles and designate these as chromosomes. A 120 kb chromosome was mapped in detail. It is shown to contain the three ribosomal genes (rrn26, rrn18 and rrn5) plus two genes encoding subunits of cytochrome oxidase (Cox1 and Cox3); it appears to be colinear with the 570-kb master circle map of another fertile cytoplasm (B37N) except at the "breakpoints" required to form the 120-kb circle. The presence of the 120-kb chromosome could not have been predicted by homologous recombination through any of the known repetitive sequences nor is it a universal feature of normal maize mitochondria. It is present in mitochondria of BMS suspension cultures and seedlings, but is not detectable in seedlings of B37N. No master genome was detected in BMS.