For ribosomal protein (RP) genes the start of transcription is rigidly controlled to maintain the 5′-TOP signal on the messenger RNA. The responsible regulatory mechanism is not yet fully understood. Careful comparative analysis of their proximal promoter sequences reveals common characteristics and thus provides clues to the underlying mechanism. We have extracted the proximal promoters of the 80 human cytosolic ribosomal protein genes together with the orthologous mouse sequences. After annotating the set with transcription factor binding sites based on the available literature, we searched for over-represented sequence motifs. We uncovered a novel motif that is localized at a fixed distance downstream to the transcription start. 31 out of the 80 promoters contain the motif in the same orientation around position + 62 (standard deviation 6). A second evolutionary conserved and palindromic motif is found 13 times in the RP promoter set, 9 instances of which are located upstream around position - 40. In addition, we see a characteristic profile of the GC-content and of the CpG dinucleotide frequencies. Our results support a model for the transcription of ribosomal protein genes in which the maintenance of the accurate start of transcription is provided by specific transcription factors. Such a factor binds the target DNA at a fixed location relative to the TSS, and possibly interacts directly with the basal transcription machinery. © 2005 Elsevier B.V. All rights reserved.