Fluorescent poly(lactic-co-glycolic acid) microspheres of a respirable size were fabricated for use in a fluorescent activated cell sorting assay utilizing the continuous alveolar macrophage NR8383. This is a suitable model of alveolar phagocytosis, which permitted an investigation of the influence of phospholipid structure on the inhibition of phagocytosis of microspheres. Phospholipid inhibition was found to be independent of phosphatidylcholine alkyl chain length. Head group effects were investigated by studies employing phosphatidyl-choline, -serine, and -ethanolamine, and inhibition was shown to be independent of head group. Closer modelling of the lung environment by co-culturing NR8383 on A549 alveolar epithelium showed type II secretions to also down-regulate phagocytosis. In addition, pre-incubation with microspheres coated with dipalmitoylphosphatidylcholine reduced the uptake of a second microsphere (fluorescein isothiocyanate-labelled latex).