Two different types of gap junction proteins, beta 1- and beta 2-connexin, were expressed in insect cells, either singly or together, using infection with recombinant baculovirus. Membrane fractions enriched in gap junction proteins were isolated, and connexons (hemichannels) were solubilized with detergent. These solubilized connexons were then run out on a gel filtration column which was capable of partially separating the two homomeric connexons. It was found that connexons from cells co-infected with both types of baculovirus ran together on this column, whereas connexons from cells infected separately and mixed before solubilization did not, suggesting that in the co-infected cells the two types of connexin are assembled into heteromeric hemichannels.