Uptake of the fluorescent probe, 1-anilino-8-naphthalene-sulfonate (I) into isolated rat liver cells was studied using both fluorescence and filtration methods. The time course of the fluorescence enhancement of I after addition to the isolated liver cells was analyzed in terms of rapid, medium, and slow phases. The slow phase (half-time approximately 7 min) was characteristic of viable cells. The fluorescence enhancement was proportional to the amount of I taken into the cells, as measured by the filtration method. The uptake of I followed Michaelis-Menten kinetics with an apparent Km of 39 microM and Vmax of 1.4 nmole/10(6) cells/min. The temperature coefficient (Q10) of the uptake of I was found to be approximately 1.9. No pH optimum was observed, and various metabolic inhibitors did not affect the uptake of I. Among the amino acid reagents used, only 2,4-dinitrofluorobenzene decreased the uptake of I (by approximately 45%). The effects of various organic anions on the uptake of I were measured. The inhibition of the uptake of I by sulfobromophthalein could be analyzed in terms of competitive inhibition; the slight inhibition by sodium taurocholate could not. It is concluded that the uptake of I is a carrier-mediated facilitated process, and that the carrier is common to both I and sulfobromophthalein.