A gene encoding a single-chain antibody (scFv) which specifically binds the epidermal growth factor receptor (EGFR) has been constructed from hybridoma cells producing the R1 monoclonal antibody. The gene, designated scFv-R1R, was introduced into EGFR transformed NIH3T3 cells via retroviral infection. scFv-R1R was directed to the lumen of the endoplasmic reticulum (ER) where it bound the extracellular domain of the receptor inhibiting its appearance on the plasma membrane. In these cells, EGF induced tyrosine phosphorylation of the EGFR and several substrates was greatly reduced. Furthermore, intracellular retention of EGFR caused a partial inhibition in the transformed growth of the cells. Intracellular expression of receptor tyrosine kinase directed scFvs is a novel approach for affecting tumor cell growth. We have recently shown that scFv-5R directed to ErbB2, another member of the ErbB family, blocks the anchorage independent growth of ErbB2 transformed cells. In order to examine the effects of scFv-R1R and scFv-5R on the long-term growth of tumor cells overexpressing either EGFR or ErbB2, retroviruses encoding the two scFvs were used to infect various human tumor cell lines. Intracellular expression of the scFvs resulted in a marked inhibition of stable colony formation in some of the cell lines. In general, inhibition was observed when overexpressed receptor was targeted. However, in some cases expression of both scFvs was incompatible with long term cell growth suggesting that heterodimers of ErbB2 and EGFR are essential for the growth of some human tumor cell lines.