The uptake of different surfactant lipids-dipalmitoylphosphatidylcholine (DPPC), phosphatidylglycerol (PG), or phosphatidylinositol (PI)-and liposomes with a surfactant-like composition by alveolar type II cells (alveolar type II cells) and macrophages (alveolar macrophages) was studied in vitro. Fluorescent-labeled liposomes containing either 86% of the studied lipid, i.e., DPPC, PG, PI, and 6% labeled phosphatidylethanolamine (PE) and 8% cholesterol or a lipid mixture similar to surfactant (DPPC, PG, PI, phosphatidylcholine, PE, and cholesterol in a weight ratio of 55:8:2:21:8:6) were incubated with alveolar macrophages and alveolar type II cells. The cell-associated fluorescence assessed by flow cytometry demonstrated a higher uptake of PG and PI by both alveolar macrophages and alveolar type II cells, and a lower uptake of DPPC by alveolar macrophages. In addition, fewer alveolar type II cells take up DPPC, whereas there are no differences for the alveolar macrophages in the number of cells involved in the uptake. Competition experiments with Texas Red-labeled liposomes and either DPPC liposomes or PI liposomes labeled with Bodipy indicated that all these liposomes are internalized via the same pathway by alveolar cells. Thus, lipid composition directly influences the (re)uptake of surfactant.