The genomes of several vertebrates contain two genes encoding proteins highly similar to threonine synthase (TS), even though the biosynthesis of l-threonine (l-Thr) is not known to occur in these animals. We report a bioinformatic analysis of the two TS-like genes, the recombinant expression of one murine TS homolog (mTSH2) and its initial biochemical characterization. Recombinant mTSH2 contained bound pyridoxal-5'-phosphate (PLP), but did not synthesize l-Thr. The enzyme did, however, bind O-phospho-homoserine (PHS; the actual TS substrate) and degraded it to alpha-ketobutyrate, phosphate, and ammonia-a known side reaction of microbial TSs. mTSH2 also degraded O-phospho-threonine (PThr) to alpha-ketobutyrate, showing that it can act as a catabolic phospho-lyase on both gamma- and beta-phosphorylated substrates. These findings suggest an unusual evolutionary origin for mTSH2, whereby an original TS enzyme became 'recycled' into a phospho-lyase upon dismissal, in metazoa, of the l-Thr biosynthetic pathway.