Transgenic hairy roots of soybean [Glycine max (L.) Merrill] induced by Agrobacterium rhizogenes support the complete life cycle of soybean cyst nematode (SCN, Heterodera glycines Ichinohe) in vitro. However, expression of SCN resistance in hairy soybean roots has not been investigated. A transgenic hairy root system would be useful in developing an assay for candidate SCN resistance genes. The objectives of this study were to characterize transgene expression in SCN-infected hairy soybean roots and to evaluate a transgenic hairy root system for investigations of resistance to SCN. Seedling cotyledons of the SCN-susceptible cultivars, Agassiz and Parker, and SCN-resistant Bell and Faribault were infected with A. rhizogenes strain K599 transformed with T-DNA binary vectors containing the gusA gene fused to promoters from either the cauliflower mosaic virus (CaMV 35S), Arabidopsis thaliana phenylalanine ammonia lyase (PAL), or bean (Phaseolus vulgaris L.) chalcone synthase-8 (CHS) genes. Nine days after inoculating transgenic hairy roots with sterile J2 nematodes, CHS-regulated β-glucuronidase (GUS) staining at infection sites increased in hairy roots of resistant Faribault and decreased in susceptible Agassiz. PAL-regulated GUS staining was absent at infection sites in hairy roots of resistant cultivars, but was increased in infection sites in susceptible cultivars. Thirty-five days after inoculation with SCN, the mean number of cysts formed on hairy roots of the resistant cultivars was about 14% of the mean number of cysts formed on hairy roots of the susceptible cultivars, indicating that the SCN resistance phenotypes were preserved in transgenic hairy roots. These results indicated that the transgenic hairy soybean root system will be useful for investigating differential transgene expression during nematode infection and evaluation of candidate SCN resistance genes.