The cause of ongoing mortality in barramundi Lates calcarifer (Bloch) in seawater culture was identified as Streptococcus iniae by biochemical and physiological tests. This is the first published record of this bacterial species in Australia and the first confirmed report of S. iniae causing mortality in barramundi. The bacterium was highly pathogenic for barramundi when challenged by bath exposure. The pathogen was found to have a LD50 of 2.5 x 10(5) and 3.2 x 10(4) colony-forming units at 48 h and 10 d respectively. Experimental challenge of barramundi resulted in high levels of mortality (> 40%) within a 48 h period. Ten days after the challenge, S. iniae could not be isolated from kidney, spleen, liver or eye of surviving fish. However, the organism was easily isolated from the brain of both moribund and healthy fish, indicating that barramundi can carry the bacterium asymptomatically.