We cloned a tumorigenic phenotype-associated cDNA encoding a tRNA synthetase-like protein from an acute-phase human myeloid leukemia cell line. The cDNA was isolated by reiterative subtraction of cDNAs synthesized from tumor-generating parental leukemia cells versus those from a nontumorigenic variant of the same cells. The selected cDNA encodes a protein that is a close homolog of one subunit of prokaryote and yeast phenylalanyl-tRNA synthetase (PheRS). The expressed protein reacts specificially with polyclonal antibodies raised against mammalian phenylalanyl-tRNA synthetase. Expression of the gene (designated CML33) was directly confirmed by Northern blot hybridization to be substantially enhanced in the tumorigenic cells compared with the nontumorigenic variant. In addition, expression of CML33 in myeloid leukemia cells was sensitive to the stage of the cell cycle and to induction of differentiation. Although the relationship between these observations and the tumorigenic state of the human myeloid leukemia cell line used in these studies is unknown, to our knowledge, this is the first demonstration in mammalian cells of tumor-selective and cell cycle stage- and differentiation-dependent expression of a member of the tRNA synthetase gene family.