The simultaneous 3D arrangement of the interphase centromeres of chromosomes 7, 11, and 17 in unstimulated human T-lymphocytes is analyzed. Using triple in situ hybridization in combination with optical sectioning and image processing, the identification of three pairs of centromeres in each nucleus and the assignment of 3D coordinates to each centromere are made. The homologous and heterologous centromere separation distance histograms are determined and compared to the hypothesized histograms for randomly distributed centromeres. The experimental nuclei are truncated spheres in shape with a principal radius of 3.7 +/- 0.3 micron and a truncated hemispherical height of 2.6 micron. None of the separation distance distributions appears to be statistically significantly different from a random model distribution.