Superparamagnetic particles were prepared with iron oxide cores of congruent to 8 nm diameter and dextran coats. After feeding the probe to the amoeba, Dictyostelium discoideum, for 15 min and chasing for 15 min, a lysosome fraction was isolated magnetically. Isolates contained 76% of ingested iron, 82% of ingested fluorescent dextran, 1.3% of cell protein, 4% of the lipid, 28% of acid phosphatase, and 5% of the vacuolar H(+)-ATPase. Enrichment in endocytic markers was congruent to 60-fold; markers for other organelles were < 0.5%. The lysosomes were homogeneous, round (0.4-1.1 microns in diameter), and frequently adherent to one another through zones of intimate apposition. Cells were also fed the iron probe continuously for 3 h to fill their entire endocytic pathway; in this case, isolates contained 3.3% of cell protein, 11% of lipid, and 49% of cell acid phosphatase. Bis(monoacylglycerol)phosphate (BMP), a lipid characteristic of lysosomes in animal cells, comprised congruent to 6% of biosynthetically labelled cell lipids and up to half of the lipid in the endocytic pathway. Essentially all of the cellular BMP was recovered in isolates prepared after 3 h of feeding. The specificity and abundance of BMP in the endocytic organelles of this early diverged protist suggests that this phospholipid serves a universal and essential function in endocytosis.