To investigate a role for surface carbohydrates in cellular malignancy, 15 different glycosylation-defective CHO cell mutants were examined for their tumorigenic and metastatic capacities after subcutaneous injection into nude mice. Most of the glycosylation mutants displayed similar or slightly decreased tumorigenicity compared with parental CHO cells. Neither parental CHO cells nor any of the mutants were observed to metastasize. However, independent isolates of one mutant type, Lec9, showed a dramatic reduction in tumor formation. The altered carbohydrates expressed at the surface of Lec9 cells appeared to be responsible for their loss of tumorigenicity, because revertants for lectin resistance were able to form tumors, and a double mutant (Lec9.Lecl) that expressed a Lecl glycosylation phenotype also formed tumors. Finally, Lec9 cells were able to form tumors in fy-irradiated nude mice, suggesting that recognition by an irradiation-sensitive host cell(s) was responsible for their reduced tumorigenicity in untreated nude mice.Transformed cell lines provide models for studying the complex events of tumor formation and metastasis. Changes in components of the plasma membrane, particularly carbohydrates, have been implicated in both phenomena. Thus, increased metastasis of B16 melanoma cells was observed when they were fused with membrane vesicles from the highly metastatic B16-F1O variant (27). Also, the extent of metastasis by a closely related group of rat mammary tumors was correlated with changes in their glycocalyx (18,19). In other studies, the level of cell surface sialylation was correlated with the metastatic potential of different murine lines (32,52,53), and many transformed cells exhibit an increased proportion of branched, N-linked cell surface carbohydrates compared with their nontransformed counterparts (1,26,50,51). Finally, several laboratories selected lectin-resistant (LecR) mutants from tumorigenic and metastatic cell lines (3,15,17,20,21,29,47,48) and found many of them to exhibit altered malignant properties. Two of these mutants express specific structural changes in their membrane carbohydrates (7,9,10). However, strict correlations between glycosylation genotype and malignant phenotype have not been reported.To establish whether cell surface carbohydrate expression plays a role in malignant properties, it is necessary to compare independent mutants of identical glycosylation genotype with revertants selected directly or generated by a second mutation that specifically alters the glycosylation phenotype induced by the first mutation. The CHO LecR glycosylation mutants previously described by this laboratory (41) offered an opportunity to pursue this approach. The glycosylation lesion expressed by each mutant affects Nlinked complex carbohydrate formation at the positions noted in Fig.