The physical distance between DNA sequences in interphase nuclei was determined using eight cosmids containing fragments of the Chinese hamster genome that span 273 kb surrounding the dihydrofolate reductase (DHFR) gene. The distance between these sequences at the molecular level has been determined previously by restriction enzyme mapping (J.E. Looney and J.L. Hamlin, 1987, Mol. Cell Biol. 7: 569-577; C. Ma et al., 1988, Mol. Cell Biol. 8: 2316-2327). Fluorescence in situ hybridization was used to localize the DNA sequences in interphase nuclei of cells bearing only one copy of this genomic region. The distance between DNA sequences in interphase nuclei was correlated to molecular distance over a range of 25 to at least 250 kb. The observed relationship was such that genomic distance could be predicted to within 40 kb from interphase distance. The correct order of seven probes was derived from interphase distances measured for 19 pair-wise combinations of the probes. Measured distances between sequences approximately 200 kb apart indicate that the DNA is condensed 70- to 100-fold in hybridized nuclei relative to a linear DNA helix molecule. Cell lines with chromosome inversions were used to show that interphase distance increases with genomic distance in the 50-90 Mb range, but less steeply than in the 25-250 kb range.