The insulin administration by pulmonary route has been investigated in the last years with good perspectives as alternative for parenteral administration. However, it has been reported that insulin absorption after pulmonary administration is limited by various factors. Moreover, in the related studies one daily injection of long-acting insulin was necessary for a correct glycemic control. To abolish the insulin injection, the present study aimed to develop a new formulation for prolonged pulmonary insulin delivery based on the encapsulation of an insulin: dimethyl-beta-cyclodextrin (INS:DM-beta-CD) complex into PLGA microspheres. The molar ratio of insulin/cyclodextrin in the complex was equal to 1:5. The particles were obtained by the w/o/w solvent evaporation method. The inner aqueous phase of the w/o/w multiple emulsion contained the INS:DM-beta-CD complex. The characteristics of the INS:DM-beta-CD complex obtained were assessed by H-1-NMR spectroscopy and Circular Dichroism study. The average diameter of the microspheres prepared, evaluated by laser diffractometry, was 2.53+/-11.8 mum and the percentage of insulin loading was 14.76+/-1.1. The hypoglycemic response after intratracheal administration (3.0 I.U. kg(-1)) of INS:DM-beta-CD complex-loaded microspheres to diabetic rats indicated an efficient and prolonged release of the hormone compared with others insulin formulations essayed.