In this study, we investigated quorum sensing regulation of virulence genes inVibrio harveyiby determining their expression levels, bothin vitroandin vivoduring infection of gnotobiotic brine shrimp. The quorum sensing master regulatorluxRand thevhpmetalloprotease showed around threefold and fivefold higher expression levels in aluxOmutant with maximum quorum sensing activity than in aluxOmutant with minimum quorum sensing activity. There was no difference in expression of thevhhhaemolysin gene between the two mutants. There was however more than 2.5-fold lower expression in an AI-2-negative mutant, suggesting that this gene is specifically regulated by AI-2 quorum sensing through a yet unknown signal transduction cascade. Thein vivoexpression data showed a peak in expression of the quorum sensing master regulatorluxRand thevhpmetalloprotease after 24 h of incubation in wild-typeV. harveyiand theluxOmutant mimicking a maximally activated quorum sensing system, whereas the expression remained low in theluxOmutant mimicking a completely inactivated quorum sensing system. Thevhhhaemolysin gene showed a peak in expression after 24 h in the wild type and a constantly low expression in an AI-2-negative mutant.