The location of centromeres and telomeres was studied in human and mouse lymphocyte nuclei (G0) employing 3D-FISH, confocal microscopy, and quantitative image analysis. In both human and murine lymphocytes, most centromeres were found in clusters at the nuclear periphery. The distribution of telomere clusters, however, differed: in mouse nuclei, most clusters were detected at the nuclear periphery, while, in human nuclei, most clusters were located in the nuclear interior. In human cell nuclei we further studied the nuclear location of individual centromeres and their respective chromosome territories (CTs) for chromosomes 1, 11, 12, 15, 17, 18, 20, and X. We found a peripheral location of both centromeres and CTs for 1, 11, 12, 18, X. A mostly interior nuclear location was observed for CTs 17 and 20 and the CTs of the NOR-bearing acrocentric 15 but the corresponding centromeres were still positioned in the nuclear periphery. Autosomal centromeres, as well as the centromere of the active X, were typically located at the periphery of the respective CTs. In contrast, in about half of the inactive X-CTs, the centromere was located in the territory interior. While the centromere of the active X often participated in the formation of centromere clusters, such a participation was never observed for the centromere of the inactive X.