Uptake of folic acid was measured in secondary cultures of skin fibroblasts from fetal rats. The cultures were made quiescent by 24 hours preincubation in medium containing 1% serum and subsequent 3 hours preincubation in phosphate buffered saline. The uptake of 3H-folic acid was linear with time during 15 seconds and reached a plateau level at 2-3 minutes. There was no further increase in the intracellular radioactivity until the end of the experiments at 10 minutes. The uptake of folic acid in fibroblasts was not concentrative and proceeded until equilibration with the extracellular concentration. Intracellular metabolic conversion of folic acid was not significant during the time of experiments (up to 10 minutes). Insulin caused a two-fold increase in the initial rate of folate uptake as determined from the 15 second uptake values. The dose response curves for the insulin effect showed that 85% of the maximal effect was exerted by 1 microM insulin. A lag period of 7-10 minutes was observed after the addition of insulin and before the effect on folic acid uptake was manifested. Thereafter the effect increased with the time of preincubation with insulin. The concentration dependence of folate uptake yielded non homogeneous curves. At low concentrations of substrate, saturable components were observed while at high concentrations (above 5 X 10(-6) M) a linear component was observed. Insulin increased the slope of the linear component and the Vmax of the saturable component while the Km remained unaltered.