AIM To investigate the influence of particle size and methoxypolyethyleneglycol (MePEG) molecular weight on the in vitro macrophage uptake and in vivo long circulating of recombinant human tumor necrosis factor-alpha (rHuTNF-alpha)-loaded stealth nanoparticles in rats. METHODS Three sizes (approximately 80, 70 and 240 nm) of poly (methoxypolyethyleneglycol cyanoacrylate-co-n-hexadecyl cyanoacrylate) (PEG-PHDCA) nanoparticles loading rHuTNF-alpha were prepared at different MePEG molecular weights (Mr 2,000, 5,000, 10,000) using the double emulsion method. The in vitro macrophage uptake and in vivo long circulating properties in rats were examined and compared. RESULTS The uptake by macrophages decreased and the half-life of rHuTNF-alpha in rat increased with the increase of MePEG molecular weight or the decrease of particle size. The linear-ships between particle size and MePEG molecular weight and the in vitro macrophage uptake and in vivo long circulating properties were fairly good. Having the highest MePEG surface density (1.32 nm(-2)) , the shortest average distance between neighboring MePEG chain (0.87 nm) and the thicker fixed aqueous layer thickness (FALT, 5.16 nm), PEG5,000-PHDCA nanoparticles (80.0 nm) earned the strongest potency of decreasing uptake by macrophages and prolonging the half-life of rHuTNF-alpha in rat. CONCLUSION Within the experimental limits, particle size and MePEG molecular weight had dramatic influence on in vitro macrophage uptake and in vivo long circulating properties of rHuTNF-alpha-loaded stealth nanoparticles.