The cells of the vertebrate lens are linked to each other by gap junctions, clusters of intercellular channels that mediate the direct transfer of low-molecular-weight substances between the cytosols of adjoining cells. Although gap junctions are detectable in the unspecialized epithelial cells that comprise the anterior face of the organ, both their number and size are greatly increased in the secondary fiber cells that differentiate from them at the lens equator. In other organs, gap junctions have been shown to play an important role in tissue development and differentiation. It has been proposed, although not experimentally tested, that this may be true in the lens as well. To investigate the function of gap junctions in the development of the lens, we have examined the effect of the gap junction blocker 18beta-glycyrrhetinic acid (betaGA) on the differentiation of primary cultures (both dissociated cell-derived monolayers and central epithelium explants) of embryonic chick lens epithelial cells. We found that betaGA greatly reduced gap junction-mediated intercellular transfer of Lucifer yellow and biocytin throughout the 8-day culture period. betaGA did not, however, affect the differentiation of these cells into MP28-expressing secondary fibers. Furthermore, inhibition of gap junctions had no apparent effect on either of the two other types of intercellular (adherens and tight) junctions present in the lens. We conclude that the high level of gap junctional intercellular communication characteristic of the lens equator in vivo is not required for secondary fiber formation as assayed in culture. Up-regulation of gap junctions is therefore likely to be a consequence rather than a cause of lens fiber differentiation and may primarily play a role in lens physiology.