FISH techniques have opened new possibilities for high-resolution genome mapping. Effective utilization of these techniques for the rapid orientation and ordering of adjacent and overlapping probes as well as for the characterization of long-range genomic contigs would facilitate physical mapping and positional cloning efforts. Here, we have evaluated our recently developed improved fiber-FISH technique for the physical mapping of a 500-kb region at 1p32 as well as for the detection of genomic rearrangement affecting this region. Our fiber-FISH technique is based on the hybridization of probes to unfixed linearized DNA fibers on a microscope slide. Preparation of the target DNA from cells embedded in pulsed-field gel electrophoresis (PFGE) blocks makes it possible to obtain long intact DNA fibers that give an excellent signal-to-noise ratio in FISH. The linear range of the method reached from 2 to 500 kb with a measuring accuracy approaching that of PFGE. Fiber-FISH was used to establish the order, orientation, and distances for several probes for this region, including six large insert phage, cosmid, and P1 clones and seven genomic subclones. This has significantly facilitated our efforts to develop a genomic contig for this region, recently discovered to contain the gene for inherited neuronal ceroid lipofuscinosis (INCL). Finally, we also demonstrated how rearrangements affecting the L-myc gene at this locus in small-cell lung cancer can be visualized with fiber-FISH. In conclusion, fiber-FISH is very useful for high-resolution physical mapping and contig evaluation as well as for detecting genetic rearrangements.