Homoserine kinase, the product of the thrB gene, catalyses an obligatory step of threonine biosynthesis. In Pseudomonas aeruginosa, unlike Escherichia coli, inactivation of the previously identified thrB gene does not result in threonine auxotrophy. A new gene, named thrH, was isolated that, when expressed in E. coli thrB mutant strains, results in complementation of the mutant phenotype. In P. aeruginosa, threonine auxotrophy is observed only when both thrB and thrH are simultaneously inactivated. Thus, thrH encodes a protein with an in vivo homoserine-kinase-like activity. Surprisingly, thrH overexpression allows complementation of serine auxotrophy of E. coli and P. aeruginosa serB mutants. These mutants are affected in the phosphoserine phosphatase protein, an enzyme involved in serine biosynthesis. Comparison analysis revealed sequence homology between ThrH and the SerB proteins from different organisms. This could explain the in vivo phosphoserine phosphatase activity of ThrH when overproduced. ThrH differs from the protein encoded by the serB gene which was identified in P. aeruginosa. Thus, two SerB-like proteins co-exist in P. aeruginosa, a situation also found in Mycobacterium tuberculosis.