Growth of temperate lettuce (Lactuca sativa) plants aeroponically in tropical greenhouses under ambient root-zone temperatures (A-RZTs) exposes roots to temperatures of up to 40 °C during the middle of the day, and severely limits root and shoot growth. The role of ethylene in inhibiting growth was investigated with just-germinated (24-h-old) seedlings in vitro, and 10-d-old plants grown aeroponically. Compared with seedlings maintained at 20 °C, root elongation in vitro was inhibited by 39% and root diameter increased by 25% under a temperature regime (38 °C/24 °C for 7 h/17 h) that simulated A-RZT in the greenhouse. The effects on root elongation were partially alleviated by supplying the ethylene biosynthesis inhibitors aminooxyacetic acid (100–500 µM) or aminoisobutyric acid (5–100 µM) to the seedlings. Application of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid to seedlings grown at 20 °C mimicked the high temperature effects on root elongation (1 µM) and root diameter (1 mM). Compared with plants grown at a constant 20 °C root-zone temperature, A-RZT plants showed decreased stomatal conductance, leaf relative water content, photosynthetic CO2 assimilation, shoot and root biomass, total root length, the number of root tips, and root surface area, but increased average root diameter. Addition of 10 µM ACC to the nutrient solution of plants grown at a constant 20 °C root-zone temperature mimicked the effects of A-RZT on these parameters but did not influence relative water content. Addition of 30 µM aminoisobutyric acid or 100 µM aminooxyacetic acid to the nutrient solution of A-RZT plants increased stomatal conductance and relative water content and decreased average root diameter, but had no effect on other root parameters or root and shoot biomass or photosynthetic CO2 assimilation. Although ethylene is important in regulating root morphology and elongation at A-RZT, the failure of ethylene biosynthesis inhibitors to influence shoot carbon gain limits their use in ameliorating the growth inhibition induced by A-RZT.