AbstractThe level of expression and cellular localization of the c‐erbB‐2 gene product in transitional cell carcinoma of the urinary tract is controversial. Analysis of the c‐erbB‐2 gene structure and comparison of its expression in the same cells by Southern, Northern and immunoblotting, and by immunocytochemistry minimize the errors of interpretation inherent in one technique. Such a ‘correlative study’ has been performed on tumours from 82 patients, c‐erbB‐2gene amplification was detected in 14 per cent of initial tumours and was associated with grade (.P<0·001). Raised levels of mRNA were seen in those tumours with increased gene copy number and in 13 per cent of the remainder. Immunoblotting detected the expected 185 kD immunoreactive protein and a 155 kD piotein associated with high gene copy number. Immunocytochemistry localized c‐erbB‐2 immunoreactivity to the cell membrane and cytoplasm, and the latter predominated. Four antibodies to c‐erbB‐2 (AB‐3, 21N, pAb 1, and NCL CB11) were compared on contiguous sections of the same tumour and showed the same pattern of immunoreactivity. Similarly, analyses carried out in three independent laboratories identified the same cellular localization. Membrane and cytoplasmic immunoreactivity was demonstrated in all tumours with gene amplification or increased mRNA levels and in 40 per cent of the remaining tumours. We showed that immunocytochemistry requires careful standardization of techniques and quantitation between different groups. However, despite variations in the intensity of immunoreactivity, the total number of positive cells remained constant. Therefore quantitation must be based on the number of positive cells and, ideally, their immunoreactive content relative to normal an