Folic acid and folates (vitamin B-9) are essential for human metabolism. Their nutritional importance mainly lies in the prevention of neural tube formation defects, and other degenerative diseases. The determination of folates in food matrices is a very complex task due to the large number of chemical compounds included under the definition of folates. In the present study, an HPLC-fluorescence detection (HPLC-FL) methodology was optimized and validated to characterize total folates in vegetables. Its application to Mediterranean wild vegetables, provide new data on folates content on these species, scarcely studied so far. The highest folates contents were found in the leaves of Rumex pulcher L., Beta maritima L. and Anchusa azurea Mill. (506, 302 and 278 mu g/100 g, respectively), as well as in the tender leaves and stems of Foeniculum vulgare Mill. and Silene vulgaris (Moench.) Garcke (272 and 268 mu g/100 g, respectively). These data show that the studied wild vegetables are mostly a very good source of folates, compared to other folate-rich plants, such as spinach, parsley or chard. These under-utilized plants deserve attention for their potential value in current diets, as they are a good alternative for the diversification of our vegetable intake and represent an important added value as rich sources of vitamin B-9.