The genomic organization of two different types of satellite DNA sequences was analysed by means of fluorescence in situ hybridization (FISH) and pulsed-field gel electrophoresis (PFGE) in barley. Satellite HvT01 was detected at all chromosome ends except the long arms of chromosomes 2 and 7. The unrelated satellite pAS1 was found at all chromosome ends except the long arm of chromosome 7 and at two interstitial sites, both located on the long arm of chromosome 4 on the standard karyotype. Southern and in situ hybridization further indicate that pAS1 also occurs interspersed in the barley genome. For most chromosome ends, the linear order of HvT01 and pAS1 could not be determined by in situ hybridization except at the short arms of chromosomes 2 and 6, where HvT01 is more distal than pAS1. This is confirmed by PFGE analysis, HvT01 being frequently associated with the telomeric repeat but not pAS1. Furthermore, we found that HvT01 occurred in clusters up to 1000 kb in size, whereas the pAS1 cluster had a maximum size of 500 kb. Sequence comparison revealed that both satellites are completely unrelated and differ considerably in their G + C contents.