Poison ivy (Toxicodendron radicans) is best known for causing exasperating allergenic delayed-contact dermatitis symptoms that last for weeks on persons who have contacted the plant. Urushiols are alkylcatechols produced by poison ivy responsible for causing this dermatitis. While urushiol chemical structures are well known, the metabolic intermediates and genes responsible for their biosynthesis have not been experimentally validated. A molecular genetic characterization of urushiol biosynthesis in poison ivy will require stable genetic transformation and subsequent regeneration of organs that retain the capacity synthesize urushiol. To this end, Agrobacterium rhizogenes was used to generate hormone-independent poison ivy hairy root cultures. Optimal conditions for hairy root formation were skotomorphic poison ivy hypocotyls prick-inoculated with A. rhizogenes, and preferential propagation of cultures with an atypical clumpy hairy root growth habit. The origin of the poison ivy accession used for A. rhizogenes prick-inoculation did not affect the initial formation of calli/ hairy root primordia, but rather significantly influenced the establishment of longterm hormone-independent hairy root growth. A. rhizogenes harboring a recombinant T-DNA binary plasmid with an intron-containing Firefly Luciferase gene produced stable transgenic hairy root lines expressing luciferase activity at high frequency. Poison ivy hairy root lines produced significantly lower steady-state urushiol levels relative to wild-type roots, but higher urushiol levels than a poison ivy undifferentiated callus line with undetectable urushiol levels, suggesting that urushiol biosynthesis requires intact poison ivy organs. The lower urushiol levels in poison ivy hairy root lines facilitated the first identification of anacardic acid metabolites initially in hairy roots, and subsequently in wild-type roots as well. This study establishes a transformation hairy root regeneration protocol for poison ivy that can serve as a platform for future reverse-genetic studies of urushiol biosynthesis in poison ivy hairy roots.