Mature-cotyledonary node transformation with Agrobacterium tumefaciens was conducted using two soybean cultivars ‘Dillon' and ‘Williams 82'. Dillon was developed by the South Carolina Agricultural Experimental Station and is maturity group VI with determinate growth habit. It is susceptible to soybean cyst nematode. Williams 82 was developed by the Illinois Agricultural Experiment Station and is maturity group III with indeterminate growth habit. It is a standard for structural and functional genomic research. In the control test, cotyledons not inoculated with Agrobacterium showed genotypic difference in response to gluphosinate exposure in vitro. Forty percent of Williams 82 cotyledons produced small green shoots, which senesced within four weeks of exposure to gluphosinate, whereas only 10% of Dillon cotyledons regenerated shoots which browned shortly after initiation. Dillon physiological responses during the transformation process indicated strong genotypic effect. Preliminary experiments established the optimal methods of inoculation, gluphosinate concentration, and exposure time for achieving transformation. In the modified procedure, one-hundred sixty-eight Dillon cotyledons were inoculated with Agrobacterium and sixteen rooted plantlets that survived the selection medium were transferred to the greenhouse. Dillon plantlets exhibited poor rooting. PCR amplification with the FLP-specific primers showed 12 plantlets had the expected 1kb FLP fragment (7% inoculated cotyledons). Out of 256 Williams 82 cotyledons, 20% regenerated shoots; however the frequency of transformation was similar to Dillon as judged by the presence of FLP fragment in rooted plantlets. Successful soybean transformation using FLP/FRT site specific recombination system can provide a useful tool for genomic modification.