In situ crystallization of rat ventricular gap junctions was accomplished by sequential dialysis of membranes against low concentrations of deoxycholate and dodecyl-beta-D-maltoside. Lipids are removed without solubilizing the protein, and the increased protein concentration in the membrane plane facilitates two-dimensional crystallization in the native membrane environment. The two-dimensional crystals have a nominal resolution of 16 A and display plane group symmetry p6 with a = b = 85 A and gamma = 120 degrees. Electron crystallography reveals that the cardiac gap junction membrane channel is formed by a hexameric cluster of protein subunits, and this hexameric design appears to be a recurring quaternary motif for the multigene family of gap junction proteins. Exposure of membranes to low concentrations of detergents may provide an approach for in situ two-dimensional crystallization of other connexins as well as other membrane proteins, especially those that are labile when solubilized as protein-detergent micelles.