We observed that chick lens-fiber gap-junction-forming proteins, connexin (Cx) 45.6 and Cx56, were associated with an unknown protein, which was then identified as major intrinsic protein (MIP), also known as aquaporin-0 (AQP0), the most abundant membrane protein in lens fibers. A 1063 bp cDNA of chick MIP(AQP0) was identified that encodes a 262 amino acid protein with a predicted molecular weight of 28.1 kDa. Dual immunofluorescence and confocal microscopy of sagittal and coronal sections of the lens tissues showed that MIP(AQP0) consistently localized with gap junction plaques formed by Cx45.6 and Cx56 during the early stages of embryonic chick lens development. Immunoprecipitation combined with immunoblotting analyses revealed that MIP(AQP0) was associated with Cx45.6 and Cx56 at these developmental stages. The specificity of this interaction was further confirmed with the silver staining of the protein components of immunoprecipitates. The pull-down analysis of lens lysates revealed that C-terminus of MIP(AQP0) probably interacted with these two fiber connexins. In late embryonic and adult lenses, however, uniform co-distribution of MIP(AQP0) and fiber connexins was largely disrupted, except for the area surrounding the actively differentiating bow regions, as was revealed by immunofluorescence and immunoprecipitation experiments. The interaction of MIP(AQP0) with lens fiber connexins in differentiating lens cells but not in mature lens fibers suggests a potential role for MIP(AQP0) in the facilitation of fiber connexins for the formation of gap junctions during lens development.