The compound rh-IFNalpha-2a-NGR can inhibit tumor angiogenesis and could be used for targeted therapy. In the present study, double antibody sandwich ELISA analysis was used to determine the concentration of rh-IFNalpha-2a-NGR in serum after intramuscular administration of various dosages to mice, rats and monkeys. The results showed that the pharmacokinetic properties of rh-IFNalpha2a-NGR after i.m. administration to mice, rats and monkeys were consistent with a one-compartment open model. The main pharmacokinetic parameters in mice (9.36 microg/kg), rats (4.68 microg/kg) and monkeys (2.34 microg/kg) after i.m. rh-IFNalpha2a-NGR were as follows: T(peak) was 0.49, 1.65 and 3.60h, C(max) was 3030.20, 654.49 and 268.13 ng/L, t1/2 was 0.39, 4.52 and 2.70 h, and AUC(0-infinity)) was 4197.65, 5784.58 and 2622.06 ng/L x h, respectively. Also, mice, rats and monkeys had their own distinct metabolic characteristics. These data would provide references for further clinical pharmacokinetic study of rh-IFNalpha2a-NGR.