Persian walnut (Juglans regia L.), which is native to Central Asia, has been widely cultivated throughout Europe over millennia as a multipurpose tree. However, only recently the naturalization process outside cultivation has been studied, mostly in temperate Europe, with no information regarding the Mediterranean region. Here we provide the irst ield investigations about the natural regeneration of walnut in two traditional Mediterranean agroecosystems: An irrigated mixed orchard in suburban areas and a non-irrigated prickly pear (Opuntia icusindica [L.] Mill.) orchard. The natural regeneration densities were statistically different in the two agroecosystems, ranging from 75 individuals per hectare in the prickly pear orchard to 200 individuals per hectare in the mixed orchard. Crows were frequently observed actively carrying walnuts in both environments. The irrigation practices and the shade provided by larger trees in the mixed orchard, and the potential beneits provided by prickly pear individuals, seemed to be crucial for seedling establishment and development in the two orchards, respectively. On the other hand, climate does not seem to represent a constraint for walnut recruitment, even if the mean annual temperature at the mixed orchard is slightly above the optimal temperature for walnut. Other biotic and abiotic factors that may trigger or hamper the naturalization process are also discussed in this paper, which explores the research needs for better understanding the naturalization potential of the Persian walnut in Mediterranean agroecosystems, as well as the effects of different land uses and future climate change on this process. ©2020 International Society of Arboriculture.