Formation of low-resistance junctions between Novikoff hepatoma cells was studied in the presence of inhibitors of protein synthesis or energy production. Cells were dissociated for 30 min in the presence of cycloheximide (CHX, 100 μg/ml), reaggregated by pelleting, and incubated for 60 min in the presence of CHX. This treatment (which inhibited protein synthesis by 99% over 30 min and prevented cell growth over 12 h) produced no significant difference in % of coupled cells or in coupling coefficient between control (0.346 ± 0.030) and treated cells (0.297 ± 0.034). Cells were treated for 12 h in CHX before dissociation and reaggregation with some decrease in % of coupled cells, but with no substantial reduction in coupling coefficient (0.298 ± 0.057). Multiple dissociations (and reaggregations) of the cells in the presence of CHX produced a decrease in 1 coupled cells and a slight reduction in coupling coefficients between treated (0.234 ± 0.029) and control preparations (0.312 ± 0.035). Cells were dissociated, treated with iodoacetate (IAA, 1 mM) for 30 min and reaggregated for 30 min. There was a small reduction in % coupled cells. Also the mean coupling coefficient was lower in treated cells (0.203 ± 0.041) compared to controls (0.393 ± 0.047), but this may reflect a general lowering of non-junctional membrane resistance. IAA resulted in a reduction of ATP to 5% of the control values. Thus, neither inhibition of protein synthesis nor severe reduction of ATP levels prevent or substantially reduce junction formation by Novikoff cells.