Markedly reduced or absent reactivity for the normally expressed A B H antigens in transitional cell carcinomas (TCCs) of the human urinary bladder appears to correlate with an aggressive clinical course. To evaluate the clinical applicability of this observation, the authors investigated the influence of methodologic variants as well as certain patient characteristics on urothelial A, B, H reactivity. Biopsy-obtained TTCs and/or normal mucosae from 104 patients were examined for the effect of fixation, paraffinization, and alcohol treatment. Variability due to the secretor status and blood group of the patient was evaluated in conjunction with methodologic factors. In the fresh-frozen state, the expected antigen was always detectable in normal mucosas but only in 69% of TCCs regardless of patient's secretor status and blood group (BG). Few normal mucosae (12%) but a large number of TTCs (46%) converted to negative after paraffin processing. Similar reduction in A, B, H reactivity was also noted when the tissues were treated with concentrations of alcohol comparable to those used in the paraffin procedure. Tumor antigens were more sensitive to alcohol. This suggested that in neoplastic cells ABH determinants are not only fewer but also disproportionately represented in polar (alcohol-soluble) molecules. Alcohol more profoundly affected tissues from nonsecretors probably because the proportion of alcohol-soluble A, B, H substances differs in secretors and nonsecretors. In the fresh-frozen state, the H antigen was detectable with the Ulex Europeus lectin in the majority of TCCs from BGO patients (79%) as well as in most tumors from BGA or B patients (90%) even in the absence of the expected A or B antigen. Alcohol treatment, however, more potently reduced reactivity for H rather than the other antigens, a phenomenon that also points to the significance of glycolipid extraction and explains the marked differences in the detectability of this antigen between fresh-frozen and paraffin-processed TCCs.