Here we describe the efficient generation of eGFP‐transgenic rats using a lentiviral approach. Analysis of the founder generation demonstrated that 46% of the offspring had stably integrated the provirus into the genome and of those 92% expressed eGFP in all blood‐derived leukocytes. In contrast to their offspring, all founder rats were mosaic with regard to eGFP‐expression, suggesting delayed viral transduction after injection. The expression level of eGFP in the F1 generation is influenced by and segregates with the site of proviral integration. Interestingly, a single copy of the transgene is sufficient for reliable detection by flow cytometry, irrespective of the leukocyte subtype analyzed. Adoptive transfer of purified CD4⁺ T‐lymphocytes from transgenic rats and subsequent reisolation from various organs further demonstrated that expression of the lentiviral transgene is maintained in a foreign host and therefore allows for efficient tracking of transferred cells. Taken together, lentivirally generated eGFP‐transgenic rats are a powerful tool for various applications in immunology and presumably also many other fields. genesis 39:94–99, 2004. © 2004 Wiley‐Liss, Inc.