Gene organization and functional motif analyses of the 123 two-component system (2CS) genes in Pseudomonas aeruginosa PAO1 were carried out. In addition, NJ and ML trees for the sensor kinases and the response regulators were constructed, and the distances measured and comparatively analyzed. It was apparent that more than half of the sensor-regulator gene pairs, especially the 2CSs with OmpR-like regulators, are derivatives of a common ancestor and have most likely co-evolved through gene pair duplication. Several of the 2CS pairs, especially those with NarL-like regulators, however, appeared to be relatively divergent. This is supportive of the recruitment model, in which a sensor gene and regulator gene with different phylogenetic history are assembled to form a 2CS. Correlation of the classification of sensor kinases and response regulators provides further support for these models. Upon comparison of the phylogenetic trees comprised of sensors and regulators, we have identified six congruent clades, which represent the group of the most recently duplicated 2CS gene pairs. Analyses of the congruent 2CS pairs of each of the clades revealed that certain paralogous 2CS pairs may carry a redundant function even after a gene duplication event. Nevertheless, comparative analysis of the putative promoter regions of the paralogs suggested that functional redundancy could be prevented by a differential control. Both codon usage and G+C content of these 2CS genes were found to be comparable with those of the P. aeruginosa genome, suggesting that they are not newly acquired genes.