It was found that AH66, a rat ascites hepatoma inherently refractory to vincristine, exhibited definite resistance to a different class of antitumor agents- adriamycin and actinomycin D. In comparative studies with AH13, a sensitive strain of rat hepatoma, significantly lower levels of cellular retention as well as uptake of these drugs were observed with AH66 cells. However, in the presence of 2, 4-dinitrophenol in glucose-free medium, the uptake of vincristine by AH66 cells was remarkably increased, and addition of glucose induced a spontaneous efflux of drug that had been taken up. These results, in good accord with those for a subline of P388 mouse leukemia which acquired resistance to either adriamycin or vincristine, suggest that there exists an active efflux system common to these different kinds of antitumor agents in naturally resistant rat tumor cells. Further studies revealed that Tween 80 also enhanced the uptake of vincristine by AH66 cells by interfering with the efflux of this drug and thus partially restored the sensitivity in vitro of AH66 cells to vincristine.