We have examined cell clones obtained from a 13762 mammary adenocarcinoma tumor and its spontaneous lung metastasis for phenotypic stability during serial culture passage in vitro. Two clones that varied markedly in their metastatic properties were chosen for further examination. One of these clones (MTC) obtained from the parental transplanted tumor initially failed to metastasize within 23 days post-injection s.c. but gained the ability to form spontaneous pulmonary metastases after several serial passages in vitro. Another clone (MTLn3) derived from a spontaneous lung metastasis was initially higher metastatic from short-term culture, but lost the potential to form large numbers of spontaneous lung metastases with long-term culture. In contrast to MTA, clone MTLn3 displayed lymph-node metastasis, and the frequency of lymph-node involvement increased when late-passage cultures of MTLn3 cells were assayed in vivo. Both clones from late-passage cultures produced larger tumor sizes at the primary (mammary fat pad) injection sites compared to early passage cells. The morphologies of MTC cells changed with serial tissue culture passage, while the morphologies of MTLn3 cells did not change. The display of fibronectin on MTC cells by immunofluorescence did not change with culture passage; fibronectin was not detected in cultures of clone MTLn3. Fibronectin was also found on MTC cells by cell surface labelling using lactoperoxidase-catalyzed iodination-sodium dodecylsulfate polyacrylamide gel electrophoresis-autoradiography. Iodination of fibronectin on MTC cells did not vary with culture passage, and as in immunofluorescence experiments it was not detected on MTLn3 cells. There was a decrease in exposure of certain cell surface proteins on MTC cells with culture passage, but we did not detect modifications with this procedure that correlated with culture passage of MTLn3 cells. We conclude that prolonged culture in vitro can result in modifications fo metastatic and cell-surface properties of tumor cell clones.