Loss, deletion or rearrangement along large portions of the long arm (q- arm) of chromosome 6 occurs in > 80% of late-stage human melanomas, suggesting that genes controlling malignant characteristics are encoded there. Metastasis, but not tumorigenicity, was completely suppressed in the human melanoma cell line C8161 into which an additional intact chromosome 6 had been introduced by microcell-mediated chromosome transfer. Our objective was to refine the location of a putative metastasis suppressor gene. To do this, we transferred an intact (neo6) and a deletion variant [neo6qdel; neo6(del)(q 16.3-q23)] of neomycin-tagged human chromosome 6 into metastatic C8161 subclone 9 (C8161.9) by MMCT. Single cell hybrid clones were selected in G-418 and isolated. Following verification that the hybrids retained the expected regions of chromosome 6 using a panel of polymorphic sequence-tagged sites, the hybrids were tested for tumorigenicity and metastasis in athymic mice. As reported previously, intact, normal chromosome 6 suppressed metastasis whether tumor cells were injected i.v. or into an orthotopic (i.e., intradermal) site. In contrast, metastasis was not suppressed in the neo6qdel hybrids. Tumorigenicity was unaffected in hybrids prepared with either chromosome 6 donor. These data strongly suggest that a human melanoma metastasis suppressor locus maps between 6q16.3-q23 (κ40 cM). (C) 2000 Wiley-Liss, Inc.