In Drosophila, several genetic phenomena are most easily explained by a model in which homologous chromosomes pair, at least transiently, and use regulatory information present on only one homolog to pattern expression from both homologs [1] [2] [3]. To accomplish pairing of sites on different chromosomes, there must be a mechanism by which communication between homologs is facilitated. However, except in the case of meiotic prophase, directed, rapid chromosomal movement has not yet been observed. Some studies suggest that chromosomes are relatively immobile during interphase [4] [5], while others suggest that chromatin can reposition during interphase [6] [7] [8] and may be free to undergo substantial Brownian motion [9]. Using high-resolution, three-dimensional imaging techniques, we determined directly the structure and nuclear location of eleven different loci, both active and inactive, in embryos at cycle 14, the mid-blastula transition. We show that during a single interphase, portions of chromosomes moved in a cell-cycle-specific, directed fashion, independently and over long distances. All eleven regions showed movement, although the genes closer to the centromere moved faster (0.7 microm/minute) and over long distances (5-10 microm), whereas those nearer the telomere expanded in the same place and became oriented along the nuclear axis. Gene motion was independent of replication, transcription and changes in nuclear shape. Because individual genes on the same chromosome move independently, the movement is unlikely to be mediated by centromeres, Brownian motion or random drift and must be caused by an active mechanism.