Evidence in support of the C₂ oxidative photosynthetic carbon cycle stems from several types of experiments. Using mutant Arabidopsis plants lacking phosphoglycolate phosphatase (the enzyme that catalyzes reaction 2 in textbook Table 8.2), C. Somerville and W. L. Ogren demonstrated that radioactivity from ¹⁴CO₂ accumulates in 2-phosphoglycolate under conditions favoring photorespiration (high concentrations of O₂, low concentrations of CO₂), but not under nonphotorespiratory conditions (low O₂, high CO₂). In addition, glycolate synthesis in vivo is dependent on O₂ and is inhibited by CO₂ in a competitive manner, as expected from the primary role of rubisco in the cycle.

Several studies have shown that, as with the Calvin cycle, the enzymes of the C₂ oxidative photosynthetic carbon cycle catalyze all the proposed reactions in vitro at rates higher than the in vivo rate of photorespiration. In addition, the participating enzymes are localized in the choroplast (rubisco, phosphoglycolate phosphatase, glycerate kinase), the peroxisome (glycolate oxidase, catalase, hydroxypyruvate reductase, and the two aminotransferases), and the mitochondrion (glycine decarboxylase and serine hydroxymethyltransferase) (see textbook Table 8.2) (Hatch and Osmond 1976; Tolbert 1981).