The uptake of [3H]choline was investigated using isolated perfused rat lungs and primary cultures of granular pneumocytes isolated by tryptic digestion of rat lungs. Metabolic products were separated from free choline by chloroform:methanol extraction and column chromatography. Tissue-associated [3H]choline increased progressively in the perfused lung, and estimated mean intracellular concentration at 2 h was 12 times the extracellular concentration (5 microM). Choline uptake was inhibited by ventilation with CO and by perfusion with the choline analog, hemicholinium-3 (HC-3). Isolated granular pneumocytes also accumulated choline against a concentration gradient by an energy-dependent process. The concentration for half-maximal uptake, after correction for the diffusion component, was estimated at 18 +/- 4 microM (mean +/- SE; n = 3), and the estimated maximal rate of uptake was 213 +/- 44 pmol/min/microliter cell water. HC-3 inhibited uptake by approximately 50% at a concentration of 10(-4) M. There was no effect on uptake when Na+ in the medium was replaced by Li+ or N-methylglucamine+. These results indicate that granular pneumocytes possess a transport system that results in accumulation of choline against a concentration gradient. The characteristics of uptake indicate that this system is similar to the low affinity choline transport system of other organs.