Stable carbon isotope signatures are often used as tracers for environmentally driven changes in photosynthetic δ(13)C discrimination. However, carbon isotope signatures downstream from carboxylation by Rubisco are altered within metabolic pathways, transport and respiratory processes, leading to differences in δ(13)C between carbon pools along the plant axis and in respired CO(2). Little is known about the within-plant variation in δ(13)C under different environmental conditions or between species. We analyzed spatial, diurnal, and environmental variations in δ(13)C of water soluble organic matter (δ(13)C(WSOM)) of leaves, phloem and roots, as well as dark-respired δ(13)CO(2) (δ(13)C(res)) in leaves and roots. We selected distinct light environments (forest understory and an open area), seasons (Mediterranean spring and summer drought) and three functionally distinct understory species (two native shrubs-Halimium halimifolium and Rosmarinus officinalis-and a woody invader-Acacia longifolia). Spatial patterns in δ(13)C(WSOM) along the plant vertical axis and between respired δ(13)CO(2) and its putative substrate were clearly species specific and the most δ(13)C-enriched and depleted values were found in δ(13)C of leaf dark-respired CO(2) and phloem sugars, ~-15 and ~-33 ‰, respectively. Comparisons between study sites and seasons revealed that spatial and diurnal patterns were influenced by environmental conditions. Within a species, phloem δ(13)C(WSOM) and δ(13)C(res) varied by up to 4 ‰ between seasons and sites. Thus, careful characterization of the magnitude and environmental dependence of apparent post-carboxylation fractionation is needed when using δ(13)C signatures to trace changes in photosynthetic discrimination.