Leaf material and seedlings of species representing all gymnosperm orders were tested for 1‐aminocyclopropane‐1‐carboxylate (ACC) oxidase activity. Seedlings of Pinus nigra, Pinus radiata, Pseudotsuga menziesii, Cupressocyparis leylandii, Ephedra major and E. nevadensis showed high level in vitro ACC oxidase activity. The enzyme from seedlings of Pinus nigra var. nigra (Arnold) was shown to resemble the angiosperm enzyme in a requirement for ascorbate, carbon dioxide and Fe(II). In contrast, seedlings of Ginkgo biloba, Dioon edule, Zamia furfuraceae and Cycas revoluta showed no detectable ACC oxidase activity. Leaf material from species representing all orders of gymnosperms was also tested for ACC oxidase activity in vitro, but none could be demonstrated. The results presented here support an origin of ACC oxidase in a common ancestor of the angiosperms, Gnetales and Coniferales.