A physical map of the Mycoplasma genitalium genome has been prepared using pulsed-field gel electrophoresis. This report details recent efforts made to add markers or specific loci to this map in the absence of any mutants or system of genetic exchange. A total of 44 random clones were partially sequenced. Computer analysis was performed in an attempt to identify homologies with genes already recorded in the DNA sequence database. Clones with a large extent of homology to genes from other microorganisms have been assigned to specific loci on the M. genitalium map by hybridization to selected restriction digests. The additional data has facilitated an updated version of the physical map, and verified this random sequencing method as a useful mapping procedure as well as offering new insight into the physiological processes of this fastidious organism.