SUMMARYChinese hamster Wg3-h-o cells which were descended from DON cells have been mutagenized and selected for derivatives defective in metabolic cooperation via gap junctions (i.e., met-). The selection protocol included four consecutive cycles of cocultivating mutagenized cells, deficient in hypoxanthine phosphoribosyltransferase (HPRT) and wild-type cells in the presence of thioguanine (cf Slack, C, Morgan, R H M & Hooper, M L, Exp cell res 117 (1978) 195-205) [8]. We carried out the last two selection cycles in the presence of 1 mM dibutyryl cyclic adenosine monophosphate (db-CAMP). The isolated Chinese hamster CI-4 cells which expressed the met-phenotype most stringently showed the following characteristics:1. In standard culture medium no cell-cell coupling was detected among CI-4 cells when assayed by injections of the fluorescent dye Lucifer yellow or by electrical measurements. Between 73 and 100% of the met+ parental cells were coupled under these conditions. Up to 14% positive contacts were found between CI-4 cells and Chinese hamster Don cells (met'). Confluent CI-4 cells grown in the presence of 1 mM db-CAMP showed 9% coupled cells.2. No gap junction plaques were found on electron micrographs of freeze-fractured, confluent CI-4 cells. The met+ parental cells showed small gap junction plaques (0.013 % of the total cell surface analyzed).3. CI-4 cells exhibited 16% positive contacts and the parental Wg3-h-o cells showed 92 % positive contacts in autoradiographic measurements of metabolic cooperation with DON cells. On an extracellular matrix, prepared from normal embryonic libroblasts, metabolic cooperation between CI-4 and DON cells was autoradiographically measured to be 68 %. Other cells of spontaneous met-phenotype (for example mouse L cells or human fibrosarcoma HT1080 cells) also appeared to exhibit increased metabolic cooperation when grown on an extracellular matrix and assayed by autoradiographic measurements. When tested by Lucifer yellow injections, however, only very few positive contacts were found for CI-4/DON cell pairs and no positive contacts were found among mouse L cells grown on an extracellular matrix.4. The met-defect in the genome of CI-4 cells was cured in somatic cell hybrids with mouse embryonic fibroblasts or with mouse embryonal carcinoma cells. The results of isozyme and karyotype studies of met-, as well as met+ somatic cell hybrids suggest that mouse chromosome 16 may be involved in complementation of the met-defect.Most cells in organs and in tissue culture show cell-cell coupling via gap junctions which-can be demonstrated by the spreading of electrical signals, injected fluorescent dyes or radioactively labelled metabolites between contiguous cells [l, 21. In many cases gap junction plaques, i.e., arrays of particles of polygonal symmetry, have been demonstrated by different techniques of electron microscopy on apposed membranes of contiguous cells. Since the appearance of gap junction plaques and the demonstration of cell-cell coupling (by one or more of