Ostriches were inoculated with a highly pathogenic avian influenza (HPAI) virus of ratite origin, A/emu/Texas/39924/93 (H5N2) clone c1B. The aim of this study was to evaluate the pathogenicity of this isolate for ostriches and to assess the ability of routine virologic and serologic tests to detect infection. Avian influenza virus (AIV) was isolated from tracheal swabs from 2 to 12 days postinfection and from cloacal swabs from 3 to 10 days postinfection. AIV was also isolated from a wide range of tissues. Birds seroconverted as early as 7 days postinfection. This study indicates that HPAI virus of ratite origin replicates extensively in infected ostriches without causing significant clinical disease or mortality.