The water use efficiency, growth and anatomic-physiological parameters of Mediterranean medicinal xerophytes on a green roof was investigated, under the effect of water stress and different types of substrate. Rooted cuttings of Convolvulus cneorum, Origanum dictamnus, Sideritis athoa, Atriplex halimus and Lomelosia cretica were planted on a roof in Athens, Greece. Two substrate types, 10 cm deep, were used, i.e. grape marc compost: perlite: soil: pumice (3:3:2:2, v/v) and a lighter one, i.e. grape marc compost: perlite: pumice (3:3:4, v/v). Two irrigation frequencies were applied during the dry period (summer), normal and sparse, when substrate moisture was 17-20% and 5-11%, respectively. Water use efficiency was increased by sparse irrigation in A. halimus and L. cretica and by soil substrate in C. cneorum. Sparse irrigation reduced dry weight in O. dictamnus, S. athoa and L. cretica and in A. halimus in combination with soilless substrate. It increased leaf thickness in all species and reduced Chlολ in all species, other than S. athoa, while in C. cneorum, O. dictamnus and L. cretica this was evident only in the presence of soil in the substrate. In summer, one d before irrigation, stomatal resistance (Rleaf) was increased and maximum quantum yield of PSII (ΦPSIIο) decreased indicating water limitation, while both parameters were restored to normal levels one d after irrigation indicating that both can serve as sensitive indicators of the onset of water stress. ΦPSIIο values, never fell to critical levels, excluding irreversible damage to the photosynthetic apparatus. Therefore, all five plant species are recommended for use on extensive green roofs in semi-arid areas.