A high-resolution fluorescence methodology for nonisotopic in situ hybridization was applied to determine the positions occupied by several single-copy genes, DNA sequences, and integrated viral genomes on sister chromatids. The lateral and longitudinal mapping of the probes was performed on prometaphase and metaphase chromosomes. A fixed lateral position, exterior or median in relation to the longitudinal axis of the chromatids, was observed for a given probe, with a symmetrical position of the double fluorescent spots. This position appears to be independent of chromosome condensation stage from prometaphase to metaphase. These observations suggest an opposite helical-handedness conformation of DNA on both chromatids with a mirror symmetry. They support the model of chromosome packaging recently proposed by Boy de la Tour and Laemmli. Moreover, our results indicate that the last stages of chromosome condensation occur by packing down the coils without further coiling.