To elucidate the relationship between the stimulation of Na+ and K+ fluxes by thyroid hormone and the induction of the Na,K-ATPase, we performed a detailed comparison of the time courses of these hormonal effects in a rat liver cell line, clone 9. Stimulations of passive K+ efflux, passive Na+ influx, and ouabain-inhibitable K+ uptake were all evident within 6-12 h of exposure of cells to T3 (10(-7) M). The time course of the induction of Na,K-ATPase activity closely paralleled that of the increase in the rate of Na+ and K+ fluxes. The maximal stimulatory effects of T3 on ouabain-inhibitable K+ uptake and Na,K-ATPase activity at 72 h were +49% and +36%, respectively. Intracellular Na+ and K+ contents were virtually unchanged during these increases in ion fluxes and Na,K-ATPase activity, suggesting an efficient homeostatic adaptation to the augmented passive "leak" of Na+ and K+ down their transmembrane concentration gradients. T3 treatment for 72 h was also shown to stimulate both lactate production (+62%) and [3H]2-deoxyglucose uptake (+82%) in these cells. The onset of these effects appeared to precede that of the stimulation of Na+ and K+ fluxes, being detectable at 4 h. Neither these latter effects of T3 nor the stimulation of ouabain-inhibitable K+ uptake could be demonstrated when RNA or protein synthesis was inhibited by actinomycin D or cycloheximide, respectively. It is concluded that in clone 9 cells thyroid hormone causes increases in passive Na+ influx, passive K+ efflux, active Na,K transport, and Na,K-ATPase activity whose time courses are closely parallel.