Insulin and insulin/poly(ethylene glycol) (PEG)‐loaded poly(l‐lactide) (PLA) nanoparticles were produced by gas antisolvent (GAS) CO₂ precipitation starting from homogeneous polymer/protein organic solvent solutions. Different amounts of PEG 6000 (0, 10, 30, 50, 100, and 200% PEG/PLA w/w) or concentration of 30% PEG/PLA with PEGs with different molecular weight (MW; 350, 750, 1900, 6000, 10,000, and 20,000) were used in the preparations. The process resulted in high product yield, extensive organic solvent elimination, and maintenance of > 80% of the insulin hypoglycemic activity. Nanospheres with smooth surface and compact internal structure were observed by scanning electron microscopy. The nanospheres presented a mean particle diameter in the range 400–600 nm and narrow distribution profiles. More than 90% of drug and PEG were trapped in the PLA nanoparticles when low MW PEGs were used in the formulation, whereas the addition of high MW PEGs significantly reduced the loading yield. In all cases, in vitro release studies showed that only a little amount of drug was released from the preparations. However, formulations containing low MW PEGs allowed for a slow but constant drug release throughout 1500 h, whereas a burst was obtained by increasing the PEG MW. In conclusion, the GAS process offers a mean to produce protein‐loaded nanoparticles possessing the prerequisites for pharmaceutical applications. The PEG added to the formulation was found to play a key role in the simultaneous solute precipitation phenomena and in determining the release behavior and the chemical–physical properties of the formulation. © 2001 Wiley‐Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 90:1628–1636, 2001