Ostriches were inoculated with a laboratory-derived highly pathogenic avian influenza (HPAI) virus of emu origin, A/emu/TX/39924/93 (H5N2) clone c1B. The aim of this study was to evaluate the pathogenicity of this isolate for ostriches and assess the ability of routine virological and serological tests to detect infection. Avian influenza virus (AIV) was isolated from cloacal and tracheal swabs from 2 to 12 days post-infection. AIV was also isolated from brain, thymus, eyelid, spleen, ovary/testis, liver, air sac, proventriculum, duodenum, caecal tonsil, heart, pancreas, kidney, nasal gland and lung. Virus isolation was also possible from swabs of the luminal surfaces of the cloaca, jejunum, lower ileum, bursa of Fabricius, trachea and bone marrow. Birds seroconverted as early as 7 days post-infection. This study suggests that HPAI virus of emu origin replicates extensively in infected ostriches without causing significant clinical disease or mortality.