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Stomatal parameters (stomatal density, stomatal index and stomatal conductance) have been widely used to study vegetation response to long-term CO2 change, mostly in the Northern Hemisphere. We tested the applicability of the methods and interpretations to Australian desert vegetation, by using Eremophila deserti A. Cunn. (Myoporaceae) leaves. Subfossil samples dated at 7500 years before present and 3700 years before present from Leporillus species (stick-nest rat) middens from the Flinders Ranges were compared with herbarium and modern samples from the area. Stomatal density and stomatal conductance are problematic in their application to this species, probably because of the effect of the moisture regime on epidermal cell size. Stomatal index, which takes some account of independent variations in cell size, did allow the differentiation of long-term trends. In contrast to most other studies, these trends show an increase in stomatal index with increasing CO2, particularly over the last century. From 7500 years before present until about 1950, it is unclear whether CO2 was the most influential among a complex set of factors including different aspects of the moisture regime. In recent decades, the influence of CO2, as demonstrated statistically, accounts for most but not all the observed variation.