In addition to providing a pathway for intercellular communication the gap junction forming proteins, connexins, can serve a growth suppressive function that is both connexin and cell-type specific. To assess its potential growth suppressive function we stably introduced connexin 37 (Cx37) into connexin-deficient, tumorigenic rat insulinoma (Rin) cells under the control of an inducible promoter. Proliferation of these iRin37 cells, when induced to express Cx37, was profoundly slowed - cell cycle time increased from 2 to 9 days. Proliferation and cell cycle time of Rin cells expressing Cx40 or Cx43 did not differ from Cx-deficient Rin cells. Cx37 suppressed Rin cell proliferation irrespective of cell density at the time of induced expression and without causing apoptosis. All phases of the cell cycle were prolonged by Cx37 expression and progression through the G₁/S checkpoint was delayed resulting in accumulation of cells at this point. Serum deprivation augmented the effect of Cx37 to accumulate cells in late G₁. Cx43 expression also affected cell cycle progression of Rin cells, but its effects were opposite to Cx37 with decreases in G1 and increases in S-phase cells. These effects of Cx43 were also augmented by serum deprivation. Cx-deficient Rin cells were unaffected by serum deprivation. Our results indicate that Cx37 expression suppresses cell proliferation by significantly increasing cell cycle time by extending all phases of the cell cycle and accumulating cells at the G₁/S checkpoint.