We present the complete maps of five Mycoplasma hominis genomes, including a detailed restriction map and the locations of a number of genetic loci. The restriction fragments were resolved by field inversion gel electrophoresis or by the contour-clamped homogeneous-electric-field system of pulsed-field gel electrophoresis. All the ApaI, SmaI, BamHI, XhoI, and SalI restriction sites (total of 21 to 33 sites in each strain) were placed on the physical map, yielding an average resolution of 26 kb. The maps were constructed using three different approaches: (i) size determination of DNA fragments partially or completely cleaved with one or two restriction enzymes, (ii) hybridization analysis with purified restriction fragments and specific probes, and (iii) use of linking clones. A genetic map was constructed by hybridization with gene-specific probes for rpoA, rpoC, rrn, tuf, gyrB, hup, ftsY, the unc operon, the genes for two M. hominis-specific antigenic membrane proteins, and one gene encoding a protein with some homology to Escherichia coli alanyl-tRNA synthetase. The positions of mapped loci were partially conserved in the five strains except in one strain in which a 300-kb fragment was inverted. The numbers and order of mapped restriction sites were only partly conserved, and this conservation was restricted to certain regions. The gene order was compared with the gene order established for other bacteria and was found to be identical to that of the phylogenetically related Clostridium perfringens. The genome size of the M. hominis strains varied from 704 to 825 kb.