PNA-reactive sites, representing mainly unmasked Thomsen-Friedenreich (TF) antigen, are predictors of invasive transitional cell carcinoma. The present studies were undertaken in order: 1) to quantify the expression of PNA-reactive sites on well-characterized human urinary bladder cell lines belonging to two different transformation grades (TGr II and TGr III); 2) to identify PNA-binding glycoproteins that are restricted in their expression to tumorigenic and invasive urothelial cell lines. Flow cytometry studies revealed significant differences between TGr II and TGr III cell lines. The mean fluorescence intensities of TGr III, tumorigenic and invasive cells, were in the range of 28.4 to 57.3 arbitrary units. The TGr II cells exhibited several fold lower fluorescence intensity: 9.8 arbitrary units for HCV 29 cell line and 13.1 arbitrary units for Hu 609 cells. Neuraminidase treatment, increasing PNA-binding to TGr II as well as in TGr III cell lines, revealed the presence of cryptic PNA-binding sites. The number of cryptic PNA-binding sites seemed to be similar in TGr II and in TGr III cells and, therefore, only add to the total number of PNA-binding sites on native, untreated cells. Binding of 125I-PNA to all cellular proteins resolved by SDS-PAGE and transferred to nitrocellulose showed multiple bands. The TGr III cells lines, after desialylation, were characterized by the presence of two major PNA-binding components represented by diffuse bands with apparent molecular mass about 68-79 kDa and 116-156 kDa, respectively, and two weakly stained bands with apparent molecular mass of 51 kDa and 60 kDa. Both cell lines representing TGr II expressed high molecular mass PNA-binding component of 207 kDa. They were further characterized by the weaker staining intensity of 116-156 kDa glycoproteins as compared to TGr III cell lines.