Auxin is the mobile signal controlling the rate of growth and specific aspects of the development of plants. It has been known for over a century that auxins act as the messenger linking plant development to specific environmental changes. An often overlooked aspect of how this is accomplished is the effect of the environment on metabolism of the major plant auxin, indole-3-acetic acid (IAA). We have studied the metabolism of IAA in relation to one environmental variable, growth temperature. The model system used was an inbred line of the aquatic monocot Lemna gibba G-3, 3F7-11 grown at temperatures ranging from 5°C to 35°C. IAA levels, the rate of IAA turnover, and the patterns of label incorporation from IAA precursors were measured using stable isotope-mass spectrometric techniques and were evaluated relative to growth at the experimental temperatures. IAA levels exhibited unusually high variability in plants grown at 15°C and 20°C. Turnover rates were quite rapid throughout the range of experimental temperatures except at 25°C, where IAA turnover was notably slower. These results suggest that a transition occurred over these temperatures for some aspect of IAA metabolism. Analysis of [15N]anthranilate and [2H5]tryptophan (Trp) incorporation into IAA showed that Trp-dependent biosynthesis predominated at 15°C; however, Trp-independent biosynthesis of IAA was the major route to IAA at 30°C. The effects of growth temperature on auxin levels have been reported previously, but no prior studies correlated these effects with which pathway becomes the primary one for IAA production.