For promoter studies the cloning, subcloning and transfer to different plasmid vectors usually requires use of restriction enzymes and ligation reactions. One obstacle is the nucleotide polymorphisms of eukaryotic genomic DNA, which has the consequence that a sequence often differs from published sequences. Therefore sequencing, rigorous restriction enzyme analysis or introduction of suitable sites has to be performed prior to cloning and subcloning. In addition, conventional methods using restriction enzymes, insert purifications and ligations is expensive and labour demanding. We have developed a fast, efficient and inexpensive Cre recombinase-loxP based method, which allows cloning of promoter regions and subcloning of these into a variety of vectors in a restriction enzyme independent manner. We here demonstrate that expression of a number of reporter genes and a therapeutic gene from both a viral and 2 mammalian promoters cloned by this recombinase method have activities comparable to conventionally cloned plasmids.