The etiolated maize (Zea mays L.) shoot has served as a model system to study red light (R)-regulated growth. Previous studies have shown that R inhibition of maize mesocotyl elongation involves a change in the auxin economy. Shown here is that R causes an increased tension in the epidermis relative to the inner tissue suggesting that the growth of the epidermis is preferentially inhibited by R irradiation. This observation, taken together with previous indirect estimates of auxin within the epidermis, has prompted the hypothesis that R mediates the inhibition of mesocotyl elongation by preferentially decreasing auxin in the epidermis, a tissue which constrains the growth of the organ. We tested this hypothesis using gas chromatography-selected ion monitoring-mass spectrometry analysis of free indole-3-acetic acid (IAA) levels in both the apical 1 cm of the mesocotyl and the corresponding epidermis of etiolated and 4 hour, R-irradiated seedlings. R irradiation caused a 1.4-fold reduction in free IAA within the whole section of the apical mesocotyl. However, within the peeled mesocotyl epidermis, red light irradiation caused at least a 1.9-fold reduction in free IAA. To determine if the nearly two-fold decrease in epidermal auxin occurring after R is physiologically significant, IAA was differentially applied to opposite sides of shoots. A 2-fold difference in IAA application rate caused asymmetrical growth. Thus, the two-fold R-induced decrease in free IAA level in the epidermis, a difference sufficient to affect growth, and the rapid R-induced change in growth rate in the epidermis are consistent with the hypothesis that R causes growth of the mesocotyl to decrease by preferentially regulating the free IAA level in the mesocotyl epidermis.