The presence of cell surface receptor glycolipid, globotriaosylceramide (Gb3), is essential to confer susceptibility to the E. coli-derived verotoxin (VT). Our earlier studies showed that Gb3 is expressed in ovarian carcinoma cell lines. The Gb3 content of normal ovary, benign and malignant primary ovarian tumors, and their metastases have now been compared by verotoxin thin-layer chromatogram (TLC) overlay of the glycolipid tissue extracts. FITC-labeled VT1 B subunit binding to frozen tumor sections was also monitored histochemically. Low to undetectable levels of Gb3 were found in "normal" ovarian tissue. Gb3 was markedly increased in both benign and malignant tumors, suggesting that increased Gb3 may be related to proliferation, rather than malignancy per se. Mucinous tumors showed the least Gb3 elevation; serous tumors were variable, showing higher levels of Gb3 in less differentiated malignant tumors. By far the highest Gb3 content was observed for secondary ovarian metastases and tumors refractory to chemotherapy. Frozen sections of neoplastic ovarian tissue overlaid with fluorescein-conjugated VT1 B subunit show extensive binding to tumor cells, particularly in poorly differentiated samples and blood vessels adjacent to, and within, the tumor mass. Tumor foci were stained but stromal tissue was consistently negative both in primary tumors and metastases. VT staining of well-differentiated primary ovarian tumor sections was weak, corresponding to their low Gb3 content, but strong staining was observed in sections from a highly differentiated primary tumor from a patient who was unexpectedly refractory to clinical chemotherapy. These studies suggest that verotoxin/Gb3 targeting may provide the basis for new treatments for ovarian cancer.