We have designed a new approach to the direct cloning and rapid analysis of mammalian enhancer elements by fusing green fluorescent protein and neomycinphosphotransferase under the control of a thymidine kinase minimal promoter. DNA fragments containing known or potential enhancer elements can be inserted into a polylinker upstream of GFPneo and re-isolated from stably transfected cell lines by a direct transgene-specific polymerase chain reaction (PCR), for further analysis. C2C12 muscle cells were transfected with four vectors containing the GFPneo fusion gene regulated by the cytomegalovirus promoter, the myoD distal core enhancer and myoblast- and myotube-specific enhancers from the desmin gene. GFPneo shows robust epifluorescence by microscopy and flow cytometry and retains sufficient neo activity to permit selection of G418-resistant clones. The fluorescence signal pattern of GFPneo expressed under the control of the desmin enhancers mirrors their transcriptional profile during myogenic differentiation. This finding demonstrates the value of GFPneo as a tool to analyse differentiation stage-specific regulatory DNA elements in stably transfected mammalian cell lines. We were able to re-isolate the myoD enhancer mediating GFPneo expression from a stably transfected C2C12 clone by a transgene-specific PCR reaction, demonstrating the feasibility of using this new vector system for the isolation of regulatory sequences.