Objective To characterize green fluorescent protein (GFP) expression in the retina of the preproenkephalin-green fluorescent protein (PPE-GFP) transgenic mouse.Methods Fluorescent in situ hybridization (FISH) and immunofluorescence (IF) were first employed to confirm the successful transgenic manipulation and its application in showing retinal ENKergic neurons.Then the GFP expression was characterized using IF with antibodies to Fluorogold (FG), glial fibrillary acidic protein (GFAP) and syntaxin 1 (HPC-1).Double labeling with other retina cell markers was then performed to further clarify the neurochemical properties of GFP-expressing amacrine cells.Results (1) The FISH and IF showed a good consistency of the expressions of PPE mRNA and GFP.PPE mRNA signals were present preferentially in somata of the inner margin of the inner nuclear layer (INL), while GFP immunoreactivities were present in fibers in the inner plexiform layer (IPL) in addition to the positive somata in the INL.(2) Double immunofluorescence suggested that none of the GFP-positive cells were FG-positive ganglion cells or GFAP-positive astrocytes, while about 94% of the GFP-positive cells were HPC-1-positive amacrine cells in the INL.(3) The colocalization of GFP with several retina cell markers, such as GlyT1, GAD67, ChAT, CB and vGluT3 indicated that the GFP-positive cells in the INL were different subtypes of the amacrine cell population.(4) No GFP and PV, CR, vGluT1 or vGluT2 double-labeled neurons were observed, although the distributions of some cells were similar to the distribution of GFP.Conclusion The present results indicate the layer specific expression pattern of GFP-positive ENKergic neurons in the retina and also provided morphological evidence for further functional studies.In conclusion, the PPE-GFP mouse line can be highly useful for the study of specific retina amacrine cell population.