Atomic force microscopy has been used to characterize gap junctions isolated from the hepatopancreas of Nephrops norvegicus, The major polypeptide of these gap junctions is ductin, a highly conserved 16- to 18-kDa protein. The hydrated gap junctions, imaged in phosphate-buffered saline, appeared as membrane plaques with a thickness of 14 nm, consistent with their being a pair of apposing membranes, The upper membrane was removed by force dissection using an increased imaging force, The thickness of the lower membrane was 6 nm, giving a separation or gap between the two membranes of 2 nm, High-resolution images show fine details of the force-dissected extracellular surfaces, as previously reported for vertebrate and heart gap junctions, In addition high-resolution AFM images show for the first time detailed substructure on the cytoplasmic face of hydrated gap junctions of either vertebrate or invertebrate, The plaques had particles on their exposed and force-dissected faces. These particles were packed in a hexagonal lattice (a = b = 8.9 nm on both faces) and had a diameter of similar to 6.5 nm, with a central, pore-like depression, Fourier maps calculated from the AFM data suggested that each particle was composed of six subunits, These images show a marked similarity to the widely accepted structure of the connexon channel of vertebrate gap junctions. (C) 1997 Academic Press.