The positions of the centromeres of all 46 human chromosomes were analysed in three dimensional reconstructions of electron micrographs of 10 serially sectioned unpretreated human male fibroblast cells. The reconstructions show that the spatial positioning of the chromosomes during division is not random. The centromeres were arranged on a metaphase plate that was ellipsoidal and that tended to be flat. The distance of centromeres from the centre of the mitotic figure was correlated with chromosome size; small chromosomes tended to be central in all the metaphases. Large chromosomes were more peripheral, especially in cells that were more advanced in mitosis. Thus, there is a tendency for larger chromosomes to move outwards as metaphase advances. In many cells, the A group centromeres were overdispersed, whereas G group centromeres tended to be clustered. The acrocentric chromosomes (D and G groups) also tended to be clustered when analysed together, probably reflecting associations in nucleoli at the previous interphase. The results show that chromosome disposition is non-random and that it changes during division.