Southern blot analysis of 6 human bladder carcinoma cell lines revealed amplification of the epidermal growth factor receptor (EGFR) gene in the KU1 cell line. The amplification of the gene was about 4-fold as compared with that of human placental DNA. Several restriction endonuclease digestions revealed that there was no gross rearrangement of the EGFR gene in KU1. Northern blot analysis showed normal 10 and 5.6 kb of EGFR gene-related mRNA species. 125I-EGF binding revealed 2 distinct EGF binding sites on KU1 cells: high-affinity sites 5.7 X 10(5) receptors per cell with 1.1 nM Kd and low-affinity sites 2.3 X 10(6) receptors per cell with 7.4 nM Kd. The number of the EGFR was compatible with that of the A431 squamous carcinoma cell ine which has an amplified, rearranged and over-expressed EGFR gene. Solid-phase immuno-isolation analysis showed a single 170 kDa EGFR protein in KU1 as well as in A431. Unlike other cell lines with amplified and over-expressed EGFR gene, anchorage-dependent growth of KU1 was stimulated but not inhibited by EGF. Moreover, anchorage-independent growth of KU1 was stimulated by EGF.