Pseudomonas putida JM37 metabolized glyoxylate at a specific rate of 55 g/g dry biomass/day. In order to investigate their role, three genes encoding enzymes that are potentially involved in the conversion of glyoxylate were disrupted; namely, tartronate semialdehyde synthase (gcl), malate synthase (glcB), and isocitrate lyase (aceA). Strains with transposon insertion in either of these genes were isolated from a 50,000 clone library employing a PCR-guided enrichment strategy. In addition, all three double mutants were constructed via targeted insertion of a knock-out plasmid. Neither mutation of gcl, glcB, and aceA nor any of the respective double mutations influenced glyoxylic acid conversion, indicating that P. putida JM37 may possess other enzymes and pathways for glyoxylate metabolism.