Cytogenetic analysis of early in vitro cultures derived from human melanomas, two primary tumors (Me 10538, Me 1402) and two metastatic lesions in the same patient (Me 665/1, Me 665/2) showed non-random involvement of C-heterochromatin in clonal chromosome rearrangements. Marker chromosomes with C- and DA-Dapi-positive bands were identified in one of the metastases, Me 665/1 (m1) and in the two primary tumors, Me 10538 (m2) and Me 1402 (m3). C-positive fragments predominated in the other metastasis, Me 665/2, which lacked C-regions intercalated in rearranged chromosomes, and were also detected with appreciable frequency in the Me 665/1 and Me 1402 cells. The frequencies of marker chromosomes and their mean number per cell allowed m2 and m1 to be considered as early markers of tumor formation and m3 as a marker of tumor progression. Dissection of chromosome structure, including the origin of the intercalated C-band, has so far been achieved only with the m2 chromosome of the primary tumor Me10538. This was the only cell line which displayed few C-fragments and a narrow chromosomal distribution with a well defined mode. A gradient of malignancy could be detected in the four cell lines, by local and disseminated tumor growth in xenotransplanted mice, with the two primary melanomas 10538 and the 1402 cells at the lowest and upper extremes. This gradient closely parallels the increase in cytogenetic heterogeneity and C-heterochromatin lesions from the 10538 to the 1402 cells.