The distribution of differentially stained chromatin was studied in the Atlantic halibut (Hippoglossus hippoglossus) chromosomes (2n=48). Four pairs of homologous chromosomes were identified using a combination of traditional cytogenetic staining techniques (Giemsa/DAPI/CMA3/Ag-NO3). Chromosome 1 showed a length polymorphism (1(S)-short, 1(L)-long isoforms of the chromosome 1) which was related to the variation of the size of the Ag-NORs. In one specimen the Ag-NOR was translocated from chromosome 1 into the telomeric region on the q-arm of the chromosome 2 forming a derivative chromosome der(2)t(1(S);2)(q?;q?). Four Ag-NOR genotypes have been shown: 1(S)1(S), 1(S)1(L), 1(L)1(L) and 1(S) der(2)t(1(S);2)(q?;q?). The chromosome rearrangements did not leave any interstitially located telomeric sequences and the telomeres were confined to the ends of the chromosomes. A single chromosomal location of 5S rDNA clusters was found using the PRINS technique. In the extended metaphase spreads two adjacent clusters of 5S rDNA could be seen on one chromosome while condensed chromatin gave a single hybridization signal. Double 5S rDNA signals on the same chromosome arm suggested paracentric inversion of the minor rDNA site. 5S rDNA clusters were not co-localized with Ag-NORs. Although female and male karyotypes were compared no sex related cytogenetic markers were found.