There are little information about growth properties of low pathogenic (LP) avian influenza virus (AIV) in embryonated chicken eggs (ECEs) at different incubation temperatures. Knowledge of this information increases the quantity and quality of antigen in vaccine production process. For this purpose, 10(-5) dilution of AIV (A/Chicken/Iran/99/H9N2) was inoculated (Intra-allantoic) into 400, 11-day old specific pathogen free (SPF) ECEs in the 0.1 mL per ECE rate and incubated in 32, 33, 34, 35, 36, 37.5, 38, 39 ˚C for 72 hr in 65% humidity. Early death embryos in first 24 hr were removed. Amnio-allantoic fluid was withdrawn into the measuring cylinder, and tested for hemagglutination (HA) activity and egg infective dose 50 (EID50). The utilizable ECEs and amnio-allantoic fluid volume was significantly increased in 35 ˚C, (p < 0.05). Significant difference in HA and EID50 titers, were seen only in 39 ˚C group. Therefore, 35°C is an optimum temperature for incubation of inoculated ECEs.