Structural alterations of chromosome 6, including del(6q), are often associated with metastatic melanoma; therefore, we hypothesized that a metastasis-suppressor gene could be coded on human chromosome 6. Highly metastatic C8161 human malignant melanoma cells exhibit chromosomal changes typical of late-stage melanomas. Using microcell-mediated chromosome transfer, a copy of a normal human chromosome 6 was introduced into C8161. Three randomly selected hybrid clones (neo6/C8161.1, neo6/C8161.2 and neo6/C8161.3) were assayed for metastasis in athymic nude mice. All controls - parental C8161 cells, randomly-selected single cell clones, neo-transfected cell clones, neo11/C8161.2 and neo11/C8161.3 - were tumorigenic (270/272 mice) and metastatic (208/272 mice). neo6/C8161 hybrid cells were still tumorigenic (91/93 mice) but were not metastatic (0/195 mice). The presence of the added chromosomes was verified in cultured and tumor cells by amplification of polymorphic (CA)(n) markers using PCR-RFLP. The neo6/C8161 hybrids display growth and morphological patterns of more differentiated cells than C8161. In Northern blot analysis an inverse relationship between metastatic ability and metastasis-suppressor gene, nm23-H1, expression is observed - with clone neo6/C8161.1 expressing the highest level of nm23 transcripts, neo6/C8161.2 and neo6/C8161.3 expressing intermediate levels, and barely detectable levels are seen in C8161. Collectively, these results suggest that a malignant melanoma metastasis-regulatory gene may be located on human chromosome 6. These results further demonstrate that tumorigenicity and metastatic ability are distinct phenotypes.