Abstract. Two cotton species (Gossypium hirsutum L. cv. SJ-2 and Gossypium barbadence cv. S-5) were grown under irrigated (wet) and non-irrigated (dry) conditions in the same field. Leaf water was enriched in 18O and deuterium in the dry treatment relative to the wet treatment for both species. Only in plants of S-5 was a similar enrichment observed in leaf cellulose. In both species, the isotopic composition of leaf cellulose must reflect the isotopic composition of the actual water pool involved in cellulose synthesis. Therefore, our observations indicate that one species (SJ-2) can maintain a relative isolation of this water pool from direct evapotranspirational effects. Such plant species will more faithfully record, in the isotopic composition of organic matter, the isotopic composition of ground water. In contrast, the isotopic composition of organic matter in plants such as S-5 could be used as an integrated signal reflecting humidity conditions during growth. Water use efficiency, based on seed-cotton yield and total water applied, correlated linearly with differences in carbon isotopic ratios between species in both the wet and dry treatments and between treatments in each species.