The oocyte cell/cell-channel assay was used to identify amino acids involved in the process of cell/cell-channel formation. The expression of the rat liver gap-junction protein, connexin 32, in single oocytes, results in the accumulation of a pool of channel precursors. Upon pairing of such oocytes, cell/cell channels form rapidly from this pool. The rate of formation is affected by thiol-specific reagents and the pH. This suggests the involvement of extracellular cysteine residues in the channel formation process. Two connexin-32 mutants were generated by site-directed mutagenesis in which cysteine residues were replaced by serine. Both mutant connexins were unable to form cell/cell channels. Thus, the cysteine residues appear to play an important role in the channel formation process.