Physical maps for Frankia strains CcI3, EAN1pec and EuI1c chromosomes were constructed by the use of macrorestriction analysis and pulsed-field gel electrophoresis (PFGE). The restriction enzymes AseI, PmeI, SwaI and SspI were used to cut the Frankia chromosome into a limited number of large fragments and for double digestions. The genomes sizes, as determined by the addition of the estimated fragment sizes, were 5430 AE 35 kb, 9101 AE 109 kb and 8105 AE 842 kb for strains CcI3, EAN1pec and EuI1c, respectively. A complete physical map was achieved by the analysis of PFGE for the single and double digestions and by two-dimensional PFGE to determine doublets and overlapping fragments. For strain EuI1c, a partial genetic map was also constructed by positioning the 16S rRNA, glnII, glnA and hboO genes on the physical map. PFGE analysis of DNA with and without proteinase K treatment together with the other results suggested a circular genome.