The chloroplastidal enzyme 2-phosphoglycolate phosphatase (PGLP), PGLP1, catalyzes the first reaction of the photorespiratory C(2) cycle, a major pathway of plant primary metabolism. Thirteen potential PGLP genes are annotated in the Arabidopsis (Arabidopsis thaliana) genome; however, none of these genes has been functionally characterized, and the gene encoding the photorespiratory PGLP is not known. Here, we report on the identification of the PGLP1 gene in a higher plant and provide functional evidence for a second, nonphotorespiratory PGLP, PGLP2. Two candidate genes, At5g36700 (AtPGLP1) and At5g47760 (AtPGLP2), were selected by sequence similarity to known PGLPs from microorganisms. The two encoded proteins were overexpressed in Escherichia coli and both show PGLP activity. T-DNA knockout of one of these genes, At5g36700, results in very low leaf PGLP activity. The mutant is unviable in normal air but grows well in air enriched with 0.9% CO(2). In contrast, deletion of At5g47760 does not result in a visible phenotype, and leaf PGLP activity is unaltered. Sequencing of genomic DNA from another PGLP-deficient mutant revealed a combined missense and missplicing point mutation in At5g36700. These combined data establish At5g36700 as the gene encoding the photorespiratory PGLP, PGLP1.