Gap junction expression has been reported to control the growth of a variety of transformed cells. We undertook parallel analysis of connexins Cx32 and Cx43 in glioma cells, which revealed potential mechanisms underlying this phenomenon and led to several novel findings. Cx43, but not Cx32, suppressed C6 glioma cell growth. Paradoxically, Cx32 transfection resulted in severalfold more dye transfer than Cx43. However, Cx43 transfectants shared endogenous metabolites more efficiently than Cx32 transfectants. Interestingly, a significant portion of Cx43 permeants were incorporated into macromolecules more readily than those that transferred via Cx32. Cx43 induced contact inhibition of cell growth but in contrast to other reports, did not affect log phase growth rates. Cell death, senescence, or suppression of growth factor signaling was not involved because no significant alterations were seen in cell viability, telomerase, or mitogen-activated protein kinase activity. However, suppression of cell growth by Cx43 entailed the secretion of growth-regulatory factors. Most notably, a major component of conditioned medium that was affected by Cx43 was found to be MFG-E8 (milk fat globule epidermal growth factor 8), which is involved in cell anchorage and integrin signaling. These results indicate that Cx43 regulates cell growth by the modulation of extracellular growth factors including MFG-E8. Furthermore, the ability of a Cx to regulate cell growth may rely on its ability to mediate the intercellular transfer of endogenous metabolites but not artificial dyes.