Multiple gap junction proteins (connexins) and channels have been identified in developing and adult heart. Functional expression of the three connexins found in chick heart (connexin42, connexin43, and connexin45) by stable transfection of communication-deficient neuro2A (N2A) cells revealed that all three connexin cDNAs are capable of forming physiologically distinct gap junctions that differ in their transjunctional voltage dependence and unitary channel conductances. The transjunctional voltage dependences of connexin45 and connexin42 closely resembled those of 4-day and 18-day embryonic chick heart gap junctions, respectively. The multiple channel conductances between 80 and 240 pS, including the predominant 160 pS channel, observed in embryonic chick heart were also common to connexin42. The expression of multiple gap junction channels with distinct conductance and regulatory properties within a given tissue may account for developmental changes in intercellular communication.