A series of human chromosome 3-specific DNA fragments isolated and characterized from a lamda phage genomic library were regionally localized on human chromosome 3. This was accomplished using filter hybridization blot analysis of a human chromosome 3 hybrid cell deletion mapping panel. Twenty-three new anonymous DNA fragments were assigned to one of four physical regions of chromosome 3. Seventeen DNA fragments were mapped to the long arm of chromosome 3, including one DNA fragment that demonstrated a restriction fragment length polymorphism (RFLP). Five DNA fragments were assigned to 3p14.2----pter, including one highly polymorphic fragment sublocalized at 3p25----pter by in situ hybridization. This DNA fragment is the second reported distal 3p polymorphic probe. One DNA fragment was localized to 3p14----p14.2. In addition, three fragments previously assigned to chromosome 3 were confirmed. Polymorphic DNA probes DNF15S2 (formerly D1S1) and D3S2 were mapped to 3p14.2----pter. The previous 3p25 in situ localization of the c-raf-1 oncogene was supported by deletion panel mapping. The physical localization of these twenty-three new DNA fragments has more than doubled the number of cloned DNA fragments assigned to chromosome 3. These and future regional assignments of DNA fragment probes will facilitate construction of both a physical and genetic linkage map of chromosome 3. They may also be useful in characterizing the chromosomal and molecular aberrations involved in small-cell lung cancer (SCLC), renal cell carcinoma, other malignancies, and the 3p14.2 common fragile site.