We studied the spatial organization of chromatin in the interphase G1, S and G2 nucleus of the protozoan Trypanosoma brucei, applying in situ hybridization with conventional fluorescence and confocal scanning optical microscopy. The majority of the trypanosome telomere GGGTTA repeats from different chromosomes were found clustered together, either extending in a network through the nuclear interior or localized at the nuclear periphery. The population of one hundred mini-chromosomes was often asymmetrically located: either clustered in a narrow band in close association with the nuclear envelope or distributed into several clusters that segregated into roughly one half of the nucleus. The nuclear organization may undergo modifications during the cell cycle and development. We conclude that non-random spatial positioning of DNA exists in the nucleus of this protozoan. Finding a high level of structural organization in the interphase nucleus of T.brucei is an important first step towards understanding chromosome structure and functioning and its role in the control of gene expression.