Gene constructs containing the Cauliflower mosaic virus (CaMV) 35S promoter and a sequence coding either for a green fluorescent protein (GFP) or for firefly luciferase were transfected into Chinese hamster ovary (CHO) cells. Both reporter genes were expressed to significant levels. The 35S promoter was 40 times less active than the human eF1α promoter, which is known to be one of the most potent promoters in mammalian cells. The 35S promoter must therefore be considered to be a promoter of significant potency in mammalian cells. RT-PCR analysis suggested that transcription initiation in CHO cells occurred between the TATA box and the transcription start site of the 35S promoter that function in plant cells. Further analysis by 5’RACE confirmed that transcription was initiated in CHO cells at different sites located essentially between the TATA box and the plant transcription start site, showing that 35S promoter activity in animal cells is due to the presence of promoter elements that are functional in mammalian cells, but that are not those used in plants. The data reported here raise the possibility that genes controlled by the 35S promoter, which is commonly used in transgenic plants, have the potential for expression in animal cells.