Trypanosoma brucei contains two tandemly arranged genes for glycerol kinase. The downstream gene was analysed in detail. It contains an ORF for a polypeptide of 512 amino acids. The polypeptide has a calculated molecular mass of 56 363 Da and a pI of 8.6. Comparison of the T. brucei glycerol kinase amino-acid sequence with the glycerol kinase sequences available in databases revealed positional identities of 39.0-50.4%. The T. brucei glycerol kinase gene was overexpressed in Escherichia coli cells and the recombinant protein obtained was purified and characterized biochemically. Its kinetic properties with regard to both the forward and reverse reaction were measured. The values corresponded to those determined previously for the natural glycerol kinase purified from the parasite, and confirmed that the apparent Km values of the trypanosome enzyme for its substrates are relatively high compared with those of other glycerol kinases. Alignment of the amino-acid sequences of T. brucei glycerol kinase and other eukaryotic and prokaryotic glycerol kinases, as well as inspection of the available three-dimensional structure of E. coli glycerol kinase showed that most residues of the magnesium-, glycerol- and ADP-binding sites are well conserved in T. brucei glycerol kinase. However, a number of remarkable substitutions was identified, which could be responsible for the low affinity for the substrates. Most striking is amino-acid Ala137 in T. brucei glycerol kinase; in all other organisms a serine is present at the corresponding position. We mutated Ala137 of T. brucei glycerol kinase into a serine and this mutant glycerol kinase was over-expressed and purified. The affinity of the mutant enzyme for its substrates glycerol and glycerol 3-phosphate appeared to be 3. 1-fold to 3.6-fold higher than in the wild-type enzyme. Part of the glycerol kinase gene comprising this residue 137 was amplified in eight different kinetoplastid species and sequenced. Interestingly, an alanine occurs not only in T. brucei, but also in other trypanosomatids which can convert glucose into equimolar amounts of glycerol and pyruvate: T. gambiense, T. equiperdum and T. evansi. In trypanosomatids with no or only a limited capacity to produce glycerol, a hydroxy group-containing residue is found as in all other organisms: T. vivax and T. congolense possess a serine while Phytomonas sp., Leishmania brasiliensis and L. mexicana have a threonine.