The dihydroxyacetone ( dha) regulon of bacteria encodes genes for the anaerobic metabolism of glycerol. In this work, genomic data are used to analyze and compare the dha regulon and related genes in different organisms in silico with respect to gene organization, sequence similarity, and possible functions. Database searches showed that among the organisms, the genomes of which have been sequenced so far, only two, i.e., Klebsiella pneumoniaeMGH 78578 and Clostridium perfringens contain a complete dha regulon bearing all known enzymes. The components and their organization in the dha regulon of these two organisms differ considerably from each other and also from the previously partially sequenced dha regulons in Citrobacter freundii, Clostridium pasteurianum, and Clostridiumbutyricum. Unlike all of the other organisms, genes for the oxidative and reductive pathways of anaerobic glycerol metabolism in C.perfringens are located in two separate organization units on the chromosome. Comparisons of deduced protein sequences of genes with similar functions showed that the dha regulon components in K.pneumoniae and C. freundii have high similarities (80–95%) but lower similarities to those of the Clostridium species (30–80%). Interestingly, the protein sequence similarities among the dha genes of the Clostridium species are in many cases even lower than those between the Clostridium species and K. pneumoniae or C. freundii, suggesting two different types of dharegulon in the Clostridium species studied. The in silico reconstruction and comparison of dha regulons revealed several new genes in the microorganisms studied. In particular, a novel dha kinase that is phosphoenolpyruvate-dependent is identified and experimentally confirmed for K. pneumoniaein addition to the known ATP-dependent dha kinase. This finding gives new insights into the regulation of glycerol metabolism in K.pneumoniaeand explains some hitherto not well understood experimental observations.