Lucifer Yellow was microinjected into Novikoff hepatoma cells and leakage was investigated under treatment with ATP (5 mM) and EGTA (5 mM) in the culture medium. In control conditions, there was no leakage in single or paired cells, except a few cases which showed very slow leakage (defined as slope < -0.0007/sec). Slow leakage rate (slope > -0.0008 but < -0.009/sec) and quick leakage rate (slope > -0.01) of intracellular dye were not seen. Dye transfer between cell pairs after Lucifer Yellow was injected into one cell was divided into two groups: quick transfer rates (4 cases, slope = -0.151 +0.0032) and slow transfer rates (15 cases, slope = -0.041 +0.0018). Under ATP treatment the intracellular dye leakage was observed in single cells (16 of 31 cases) and in cell pairs (20 of 57 cases). Extracellular low Ca2+ (EGTA treatment) enhanced the dye leakage much more: 30 of 40 cases in single cells and 21 of 36 cases in cell pairs. The leakage rates of intracellular dye under these treatments were similar to the transfer rates of the dye between cell pairs with quick and slow rates. It is suggested that the dye leakage from Novikoff cells under treatment with ATP or low [Ca2+]o shares the same mechanism as dye transfer through gap junctions, suggesting that the hemichannels in the plasma membrane can be opened under certain conditions.