Proteins present in tobacco nuclear extracts bind to a truncated cauliflower mosaic virus (CaMV) 35S promoter fragment (from -90 to +2 relative to the transcription start site) in a sequence specific manner. Gel mobility shift assays show the presence of two protein-DNA complexes that are not competed by a -47/+2 promoter fragment. DNAse I protection and DNA methylation interference reveal two protected sites in the slower migrating complex; both include the pentamer TGACG, separated by a stretch of eight nucleotides where G methylation does not prevent the binding of the proteins. The faster complex is the prevalent form at low protein concentrations. As the protein concentration increases a non-linear rise in the amount of the slower migrating complex relative to the faster one is seen suggesting that cooperative effects are involved in the binding to the second site.