Lens transparency and high refractive index presumably depend on the appropriate arrangement and distribution of lens proteins among lens fiber cells. Intercellular gap junction channels formed by alpha3 and alpha8 connexins are known to transport small molecules, ions and water, but not proteins, in the lens. Mosaic expression of green fluorescent protein (GFP) in the lens is a useful marker for monitoring macromolecule distribution between fiber cells and for constructing three-dimensional images of living lens cells. In alpha3(-/-) alpha8(-/-) double knockout (DKO) lenses, three-dimensional images of GFP-positive cells demonstrate the changes of epithelial cell surfaces and insufficient elongation of inner fiber cells. Uniform distribution of GFP between inner lens fiber cells is observed in both wild-type and alpha3(-/-) lenses. In contrast, uniform GFP distribution is slightly delayed in alpha8(-/-) lenses and is abolished in DKO lenses. Without endogenous wild-type alpha3 and alpha8 connexins, knock-in alpha3 connexin (expressed under the alpha8 gene promoter) restores the uniform distribution of GFP protein in the lens. Thus, the presence of either alpha3 or alpha8 connexins seems sufficient to support the uniform distribution of GFP between differentiated lens fiber cells. Although the mechanism that drives GFP transport between fiber cells remains unknown, this work reveals that gap junction communication plays a novel role in the regulation of intercellular protein distribution in the lens.