The more A + T rich fractionated component (FII DNA) of the Halobacterium halobium genome constitutes one third of the total DNA and upon isolation consists of covalently closed circular DNA (pHH1 and minor cccDNA) and nonsupercoiled sequences. We have investigated the physical organization of the non cccDNA in FII by a chromosome walk using one copy of the halobacterial insertion element ISH1 as a start point. This chromosome walk led to the isolation of 160 kb of chromosomal DNA containing 70 kb of FII DNA covalently linked to more G + C rich sequences (FI DNA). Copies of three previously characterized insertion elements (ISH1, ISH2, and ISH26) as well as at least 10 other repeated sequences are clustered within this chromosomal FII DNA "island". Unique sequences are found in the FI DNA flanking the FII DNA island as well as in 40 kb of FI DNA surrounding the bacterio-opsin gene. The presence of pHH1 in H. halobium and closely related species correlates with the occurrence of the characterized chromosomal FII DNA island. Halophilic purple membrane producing isolates YC81819-9, GN101, SB3 and GRA lack pHH1 and the 70 kb FII DNA, but contain all of the FI DNA sequences tested. We propose that pHH1 and this chromosomal FII DNA are characteristic genomic components of H. halobium and closely related species, and, that the 70 kb FII DNA might represent a large insertion in the chromosome of H. halobium and closely related species. The conservation of both FI and FII DNA sequences can be used for strain classification and determination of evolutionary relationships among halo-bacteria.