Endocytosis is an efficient method for transfer of genes into mammalian cells. Incorporation of adenovirus particles into gene transfer complexes greatly enhances gene delivery, probably by the release of endocytosed DNA into the cytoplasm. We report here that two different serotypes of human rhinovirus (HRV), HRV2 and HRV14, are also able to enhance receptor-mediated gene transfer. The effect of several compounds known to inhibit viral infection on HRV2- and HRV14-enhanced transfection was examined. WIN I(s) and WIN IV, two compounds which inhibit viral uncoating, had different effects on HRV2- and HRV14-enhanced gene transfer to NIH 3T3 cells. While HRV14-enhanced gene transfer was severely reduced in the presence of these compounds, virtually no effects were observed when HRV2 was used. The use of antiviral compounds thus allowed transfection of human cells, which are normally lysed rapidly upon infection with HRV. Viral activity could be mimicked by using a peptide derived from the N terminus of VP1 of HRV2. This peptide possesses pH-dependent membrane-disrupting activity and enhances gene transfer to NIH 3T3 and HeLa cells.