Murine fibroblasts, LM cells, were cultured in suspension or monolayer in a chemically defined medium without serum and exposed to polystyrene beads. The LM cells endocytized the beads in direct proportion to the bead/cell ratio and the bead surface area. However, equal volumes of beads irrespective of size or surface area were internalized. The lipid composition of the phagosome membrane differed significantly from the parent primary membrane in having higher contents of phosphatidylcholine, phosphatidylserine, and sterol but lower contents of sphingomyelin and lysophosphatidylcholine. When phagosomes isolated from suspension-cultured LM fibroblasts were exposed to trinitrobenzene-sulfonic acid at 4 degrees C, 55 +/- 1.6% of the phagosomal membrane phosphatidylethanolamine was trinitrophenylated. The asymmetric distribution of phosphatidylethanolamine across the phagosomal membrane was not affected by the bead/cell ratio, bead diameter, or exposure time of LM fibroblasts to the beads. When cells were reacted with trinitrobenzenesulfonic acid at 4 degrees C prior to phagocytosis, the amount of trinitrophenylphosphatidylethanolamine was greater in the isolated phagosomes than in the parent primary plasma membrane. Culturing LM fibroblasts in suspension or monolayer had no effect on the asymmetric distribution of phosphatidylethanolamine across primary plasma membrane bilayers. The data are consistent with the observation that LM fibroblasts grown either in suspension or monolayer internalize polystyrene beads at selective sites in the surface membrane.