Abstract Petunia hybrida is a model ornamental plant grown worldwide. To understand the HCHO-uptake efficiency and metabolic mechanism of petunia, the aseptic petunia plants were treated in HCHO solutions. An analysis of HCHO-uptake showed that petunia plants effectively removed HCHO from 2, 4 and 6 mM HCHO solutions. The 13C NMR analyses indicated that H13CHO was primarily used to synthesize [5-13C]methionine (Met) via C1 metabolism in petunia plants treated with 2 mM H13CHO. Pretreatment with cyclosporin A (CSA) or l-carnitine (LC), the inhibitors of mitochondrial permeability transition pores, did not affect the synthesis of [5-13C]Met in petunia plants under 2 mM H13CHO stress, indicating that the Met-generated pathway may function in the cytoplasm. Under 4 or 6 mM liquid H13CHO stress, H13CHO metabolism in petunia plants produced considerable amount of H13COOH and [2-13C]glycine (Gly) through C1 metabolism and a small amount of [U-13C]Gluc via the Calvin Cycle. Pretreatment with CSA or LC significantly inhibited the production of [2-13C]Gly in 6 mM H13CHO-treated petunia plants, which suggests that chloroplasts and peroxisomes might be involved in the generation of [2-13C]Gly. These results revealed that the C1 metabolism played an important role, whereas the Calvin Cycle had only a small contribution during HCHO metabolism and detoxification in petunia under liquid HCHO stress.