Human blood leukocytes were exposed to X rays to analyze the initial level of DNA breakage induced within different satellite DNA sequence areas and telomeres, using the DNA breakage detection-FISH procedure. The satellite DNA families analyzed comprised alphoid sequences, satellite 1, and 5-bp classical satellite DNA sequences from chromosome 1 (D1Z1 locus), from chromosome 9 (D9Z3 locus), and from the Y chromosome (DYZ1 locus). Since the control hybridization signal was quite different in each of the DNA targets, the relative increase in whole fluorescence intensity with respect to unirradiated controls was the parameter used for comparison. Irradiation of nucleoids obtained after protein removal demonstrated that the alkaline unwinding solution generates around half the amount of signal when breaks are present in the 5-bp classical DNA satellites as when the same numbers of breaks are present the genome overall, whereas the signal is slightly stronger when the breaks are within the alphoids or satellite 1 sequences. After correction for differences in sensitivity to the alkaline unwinding-renaturation, DNA housed in chromatin corresponding to 5-bp classical satellites proved to be more sensitive to breakage than the overall genome, whereas DNA in the chromatin corresponding to alphoids or satellite 1 showed a sensitivity similar to that of the whole genome. The minimum detectable dose was 0.1 Gy for the whole genome, 0.2 Gy for alphoids and satellite 1, and 0.4 Gy for the 5-bp classical satellites. Telomeric DNA sequences appeared to be maximally labeled in unirradiated cells. Thus telomeric ends behave like DNA breaks, constituting a source of background in alkaline unwinding assays.