Progression of human melanoma toward increasingly malignant behavior is associated with several nonrandom chromosomal aberrations, most commonly involving chromosomes 1, 6, 7, 9, and 10. We previously showed that introduction of human chromosome 6 into the highly metastatic human malignant melanoma cell line C8161 completely suppressed metastasis without altering tumorigenicity (Welch DR, Chen P, Miele ME, et al., Oncogene 9:255-262, 1994). Alterations of chromosome 1 are the most frequent chromosome abnormality observed in melanomas, and they frequently arise late in tumor progression. The purpose of the study presented here was to compare the effects of chromosomes 1 and 6 on malignant melanoma metastasis. By using microcell-mediated chromosome transfer, single copies of neo-tagged human chromosomes 1 or 6 were introduced into the human melanoma cell line MelJuSo. The presence of the added chromosome was verified by G banding of karyotypes, fluorescence in situ hybridization, and screening for polymorphic markers on each chromosome. The incidence and number of metastases per lung after intravenous or intradermal injection of parental MelJuSo cells was significantly (P < 0.01) greater than those of hybrids containing either chromosome 1 or chromosome 6, although chromosome 1 was a less potent inhibitor of metastasis than chromosome 6. Cultures established from primary tumors and metastases remained neomycin resistant, suggesting that portions of the added chromosomes were retained. These results strengthen the evidence for the presence of a melanoma metastasis suppressor gene on chromosome 6. neo6/MelJuSo hybrids expressed 2.4- to 3.4-fold more of the melanoma differentiation-associated gene mda-6 (previously shown to be identical to WAF1/CIP1/Sdi1/CAP20) than parental metastatic cells. mda-6/WAF1 is among the candidate genes on chromosome 6. These results also demonstrate, for the first time, the existence of metastasis suppressor genes on human chromosome 1, although these genes appear to be less potent than the one encoded on chromosome 6.