Chromosome 13 is one of the poorly mapped human chromosomes. As an example, only two cloned genes have been assigned to bands 13q22-q31. Our characterization of the critical region for the variant form of late infantile neuronal ceroid lipofuscinosis (vLINCL, locus definition CLN5) disease region on 13q22 resulted in the identification of the sequences encoding the BTF3 protein homologue gene (HGMW-approved symbol BTF3) and a novel pseudogene for RNA Helicase A (HGMW-approved symbol DDX9P). Precise visual assignment to the physical clones covering this region and the positional relationships of these genes were achieved by the use of tyramine enhancement of Fiber-FISH hybridization signals, demonstrating the power of this technique in efficient positioning of coding regions on the physical maps.