Epidermal growth factor (EGF) binds to specific high affinity receptors (EGF-Rs) and induces endosome-specific internalization and degradation of ligand-receptor complexes in lysosomes. We report here that EGF-R is down-regulated in an analogous manner during early infection of a variety of cell types by group C human adenoviruses. This effect is not a function of viral entry, nor is it due to a nonspecific increase in turnover of membrane proteins. Using a series of virus deletion mutants, the gene responsible for EGF-R down-regulation was mapped to the E3 transcription unit. The E3 gene product, a protein of MW 10,400 (10.4K), induces internalization and degradation of EGF-R, but does not affect synthesis of the EGF-R precursor. The 10.4K protein is not an EGF-like autocrine growth factor, but is similar in sequence to a region in EGF-R at the cytoplasmic face of the transmembrane domain. This suggests that down-regulation of EGF-R during adenovirus infection may occur by a novel mechanism that involves the formation of hetero-oligomers composed of 10.4K and EGF-R.