We used genetic and molecular approaches to identify mechanisms by which the gaseous plant hormone ethylene reduces lateral root formation and enhances polar transport of the hormone auxin. Arabidopsis thaliana mutants, aux1, lax3, pin3 and pin7, which are defective in auxin influx and efflux proteins, were less sensitive to the inhibition of lateral root formation and stimulation of auxin transport following treatment with the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC). By contrast, pin2 and abcb19 mutants exhibited wild-type ACC responses. ACC and indole-3-acetic acid (IAA) increased the abundance of transcripts encoding auxin transport proteins in an ETR1 and EIN2 (ethylene signaling)-dependent and TIR1 (auxin receptor)-dependent fashion, respectively. The effects of ACC on these transcripts and on lateral root development were still present in the tir1 mutant, suggesting independent signaling networks. ACC increased auxin-induced gene expression in the root apex, but decreased expression in regions where lateral roots form and reduced free IAA in whole roots. The ethylene synthesis inhibitor aminoethoxyvinylglycine (AVG) had opposite effects on auxin-dependent gene expression. These results suggest that ACC affects root development by altering auxin distribution. PIN3- and PIN7-GFP fluorescence was increased or decreased after ACC or AVG treatment, respectively, consistent with the role of PIN3 and PIN7 in ACC-elevated transport. ACC treatment abolished a localized depletion of fluorescence of PIN3- and PIN7-GFP, normally found below the site of primordia formation. These results suggest that ACC treatment increased PIN3 and PIN7 expression, resulting in elevated auxin transport, which prevented the localized accumulation of auxin needed to drive lateral root formation.