Agrobacterium tumefaciens-mediated transformation of tobacco leaves (Nicotiana tabacum) is used to study gene expression in a heterologous genetic background. Here, the Cre-loxP recombination system was used to detect T-DNA transfer by two A. tumefaciens cells harboring different binary vectors. Cre, under the control of the CaMV 35S promoter, was cloned into one vector, and a loxP cassette into another vector. A mixture of A. tumefaciens, in which each cell contained either a Cre- or loxP-vector, was co-infiltrated into tobacco leaves. After two days, excision of loxP-flanked DNA was detected by PCR and used as an estimate for co-transformation events. Strongest excision (> 50%) was observed when the loxP cassette was cloned into vector pPZP112 and Cre into pISV2678. This fast and easy technique can be used to assess the co-transformation efficiency of tobacco cells in future studies.