In plants, auxin is a key regulator of development and is unique among plant hormones in that its function requires polarized transport between neighboring cells to form concentration gradients across various plant tissues. Although putative auxin-influx and -efflux transporters have been identified by using molecular genetic approaches, a detailed functional understanding for many of these transporters remains undetermined. Here we present the functional characterization of the auxin-influx carrier AUX1. Upon expression of AUX1 in Xenopus oocytes, saturable, pH-dependent uptake of 3H-IAA was measured. Mutations in AUX1 that abrogate physiological responses to IAA in planta resulted in loss or reduction of 3H-IAA uptake in AUX1-expressing oocytes. AUX1-mediated uptake of 3H-IAA was reduced by the IAA analogs 2,4-D and 1-NOA, but not by other auxin analogs. The measured Km for AUX1-mediated uptake of 3H-IAA was at concentrations at which physiological responses are observed for exogenously added IAA and 2,4-D. This is the first report demonstrating detailed functional characteristics of a plant auxin-influx transporter. This biochemical characterization provides new insights and a novel tool for studying auxin entry into cells and its pivotal roles in plant growth and development.