Chromosome morphologies revealed by Giemsa-banded karyotypes and chromosome numbers were compared between parental tumor-, lymph node- and lung metastasis-derived rat 13762NF mammary adenocarcinoma cell lines and clones having different spontaneous metastatic potentials. Although chromosome numbers in the cell lines and clones generally correlated with DNA content by flow cytometry, ploidy did not correlate with spontaneous metastatic potentials. Chromosome number and DNA content drifted during prolonged in vitro growth in each of the cell lines and clones. Common chromosome rearrangements were found, confirming a common origin for all the cell lines and clones, and the frequency and appearance of the individual marker chromosomes fluctuated during in vitro growth. Karyotypic analyses revealed that the markers coinciding with phenotypic drift in spontaneous metastatic potential and other biological properties of parental tumor-derived clones MTC and MTF7 and lung metastasis-derived clone MTLn3 involved chromosomes 3, 4, 5, 6, and 8. Clone MTC exhibited a shift in several markers and an increase in metastatic potential at passage T20, while clone MTF7 displayed a lesser spontaneous metastatic potential at high passage (T34) concomitant with an increase in the frequency of certain marker chromosomes. Lung metastasis-derived clone MTLn3 also exhibited a shift in some marker chromosomes, colonization preference and metastatic potential to lung and lymph nodes at high tissue culture passages. The changes in marker chromosomes during in vitro passage of clones MTC and MTLn3 suggested the presence of at least two cell subpopulations which could be responsible for the observed shift in spontaneous metastatic properties. Karyotypic features of the 13762NF cell lines and clones indicate that subtle cytogenetic changes, in contrast to gross chromosomal abnormalities, may be more important in determining metastatic phenotype.