Lentiviral vectors have become attractive delivery vehicles for gene therapy investigators. Specifically, the ability of lentiviral vectors to integrate into nondividing cells and provide stable and long-term gene expression in vivo is a desirable attribute of gene therapy approaches. We report here a simple method for generating transgenic mice using lentiviral vectors, which could be useful models for gene therapy. After removal of the zona pellucida, fertilized eggs were co-incubated with oncoretroviral or lentiviral vectors. The resulting blastocysts were transferred into uteri of pseudo-pregnant females. In both cases, around 60-70% of founder pups were transgenic as determined by PCR analysis. Southern blot analysis revealed that the transgenes were integrated at different genetic loci and transmitted through the germ line. Most of the transgenes delivered by lentiviral vectors were expressed in transgenic mice, although those delivered by oncoretroviral vectors were completely silenced. When the upstream sequences of the rhodopsin gene and the red pigment gene were used as tissue-specific promoters, consistent enhanced green fluorescent protein (EGFP) expression was observed in rod and cone photoreceptor cells, respectively, in retina. However, mice generated with the corneal epithelium-specific keratin-12 promoter displayed EGFP expression not only in cornea but also in other tissues of the mouse. We conclude that the generation of transgenic mice using lentiviral vectors is a simple and robust method to evaluate the promoter specificity in lentiviral vectors in vivo prior to undertaking a gene therapy strategy.