HeLa cells were grown in medium containing various amounts of actinomycin D for various times. Cellular localization of protein B23 was detected using an immunofluorescence technique. Bright nucleolar fluorescence was observed in untreated cells. A shifting of nucleolar to nuclear fluorescence was observed with increasing doses of actinomycin D and longer incubation times. The degree of translocation of protein B23 from nucleoli to nucleoplasm is dependent on the amount of the drug used and the duration of incubation. Short exposure (0.5 h) of HeLa cells to actinomycin D (0.01-0.25 microgram/ml) induced "reversible" translocation of protein B23, inhibition of cell growth, and RNA synthesis. A majority of cells (greater than 75%) treated with actinomycin D (0.01-0.25 microgram/ml) for 0.5 h still retained bright nucleolar fluorescence. A shifting of nucleolar to nuclear fluorescence as well as inhibition of cell growth and RNA synthesis were observed within 6 h after the removal of the drug. However, at the extended periods (greater than 24 h) after drug removal, RNA synthesis and cell growth resumed at the normal rate, and protein B23 relocated from nucleoplasm to nucleoli. This is in contrast to the results obtained from the experiments using higher doses (1 microgram/ml; 0.5 h) or longer (0.25 microgram/ml; 2 h) exposure of HeLa cells to actinomycin D, which induced irreversible B23 translocation as well as irreversible inhibition of cell growth and RNA synthesis. These results indicated that actinomycin D can be a reversible inhibitor depending on the drug extracellular concentrations and exposure times. Our results also indicated that "B23 translocation" is closely associated with states of cell growth and inhibition of RNA synthesis. "B23 translocation" may therefore be a simple and rapid method for assessing the inhibition of cell growth in response to antitumor therapy.