Rats bearing Novikoff hepatoma ascites cells were given i.p. injections of actinomycin D, doxorubicin, or daunorubicin. Four hours after injection, tumor cells were removed from the ascites fluid and analyzed for protein B23 translocation using an immunofluorescence technique. Bright nucleolar fluorescence was observed in untreated cells. Treatment with actinomycin D (1.25 mg/kg), doxorubicin (25 mg/kg), or daunorubicin (12.5 mg/kg) produced a uniform nucleoplasmic fluorescence. This change in immunofluorescence distribution indicated that protein B23 translocated from the nucleolus to the nucleoplasm after drug treatment. These results are an extension of previous studies with HeLa cells (Yung et al., Cancer Res. 46: 922-925, 1986). Doxorubicin-resistant and -sensitive mouse leukemia cells (P388) were cultured in medium containing various doses of doxorubicin for 4 h, and the responsive levels of the cells to doxorubicin were compared. At 50 micrograms/ml doxorubicin, 86% of the doxorubicin-sensitive cells showed uniform nucleoplasmic fluorescence, and less than 2% of the cells retained nucleolar fluorescence. At this same dose, only 9% of the resistant cells showed nucleoplasmic fluorescence, and 75% of the cells retained nucleolar fluorescence. At 100 micrograms/ml, about 26% of the resistant cells showed translocation, in contrast to 100% of the sensitive cells that showed B23 translocation. About 57% of the resistant cells showed an intermediate effect, and about 17% of the resistant cells maintained bright nucleolar fluorescence at this dose. The resistant cells also showed less responsiveness to actinomycin D. These results suggest that identification of "B23 translocation" may be used to detect drug-resistant cells and to study the efficacy of certain antitumor agents.