In Australia, the outbreak of pandemic (H1N1) 2009 began in Melbourne, Victoria; in the first 17 days, the Victorian Infectious Diseases Reference Laboratory detected 977 cases. Although the laboratory had a pandemic plan in place, a retrospective evaluation found 3 major variations from plan assumptions: 1) higher peak demand not limited by a case definition, 2) prolonged peak demand because containment attempts continued despite widespread influenza, and 3) unexpected influence of negative test results on public health actions. Although implementation of the plan was generally successful, the greatest challenges were limited availability of skilled staff and test reagents. Despite peak demand of 1,401 tests per day, results were provided within the usual 24 hours of specimen receipt; however, turnaround time seemed slower because of slow transport times (>3 days for 45% of specimens). Hence, effective laboratory capability might be enhanced by speeding transport of specimens and improving transmission of clinical data.