The chromatin in interphase nuclei is much less condensed than are metaphase chromosomes, making the resolving power of fluorescence in situ hybridization (FISH) two orders of magnitude higher in interphase nuclei than on metaphase chromosomes. In mammalian species it has been demonstrated that within a certain range the interphase distance between two FISH sites can be used to estimate the linear DNA distance between the two probes. The interphase mapping strategy has never been applied in plant species, mainly because of the low sensitivity of the FISH technique on plant chromosomes. Using a CCD (charge-coupled device) camera system, we demonstrate that DNA probes in the 4 to 8 kb range can be detected on both metaphase and interphase chromosomes in maize. DNA probes pA1-Lc and pSh2.5.SstISalI, which contain the maize loci a1 and sh2, respectively, and are separated by 140 kb, completely overlapped on metaphase chromosomes. However, when the two probes were mapped in interphase nuclei, the FISH signals were well separated from each other in 86% of the FISH sites analyzed. The average interphase distance between the two probes was 0.50 micron. This result suggests that the resolving power of interphase FISH mapping in plant species can be as little as 100 kb. We also mapped the interphase locations of another pair of probes, ksu3/4 and ksu16, which span the Rp1 complex controlling rust resistance of maize. Probes ksu3/4 and ksu16 were mapped genetically approximately 4 cM apart and their FISH signals were also overlapped on metaphase chromosomes. These two probes were separated by an average of 2.32 microns in interphase nuclei. The possibility of estimating the linear DNA distance between ksu3/4 and ksu16 is discussed.