The ability to control multiple genes at the transcriptional level often relies on the existence of short stretches of well-defined DNA sequences, to which regulatory proteins and transcription factors bind. In this article we present a freely accessible web-based application (GRASP-DNA), that can be used to screen prokaryotic genomes for putative DNA-binding sites of a particular transcription factor or DNA-binding molecule. This application utilizes existing theories, such as information and statistical-mechanical theories, for the calculation of positive weight matrices generated from block aligned binding sites. Using these position weight matrices entire prokaryotic genomes are screened to identify sites that display a high level of sequence similarity to existing binding sites. This application can be used in combination with high-throughput technologies for gene expression analysis and binding site characterization to assist in the elucidation of global regulatory networks.