Non-photosynthetic, or heterotrophic, tissues in C₃ plants tend to be enriched in ¹³C compared with the leaves that supply them with photosynthate. This isotopic pattern has been observed for woody stems, roots, seeds and fruits, emerging leaves, and parasitic plants incapable of net CO₂ fixation. Unlike in C₃ plants, roots of herbaceous C₄ plants are generally not ¹³C-enriched compared with leaves. We review six hypotheses aimed at explaining this isotopic pattern in C₃ plants: (1) variation in biochemical composition of heterotrophic tissues compared with leaves; (2) seasonal separation of growth of leaves and heterotrophic tissues, with corresponding variation in photosynthetic discrimination against ¹³C; (3) differential use of day v. night sucrose between leaves and sink tissues, with day sucrose being relatively ¹³C-depleted and night sucrose ¹³C-enriched; (4) isotopic fractionation during dark respiration; (5) carbon fixation by PEP carboxylase; and (6) developmental variation in photosynthetic discrimination against ¹³C during leaf expansion. Although hypotheses (1) and (2) may contribute to the general pattern, they cannot explain all observations. Some evidence exists in support of hypotheses (3) through to (6), although for hypothesis (6) it is largely circumstantial. Hypothesis (3) provides a promising avenue for future research. Direct tests of these hypotheses should be carried out to provide insight into the mechanisms causing within-plant variation in carbon isotope composition.