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Abstract: β-actin has been frequently used as an internal control (gene) or as a housekeeping gene to normalize the expression of the target gene(s) or mRNA levels between different samples. However, the β-actin expression has been shown to be influenced by the sample type and experimental conditions. If β-actin could be used as a reference gene for the half-smooth tongue sole Cynoglossus semilaevis remains ill-defined. Here we evaluate the tissue-specific β-actin gene expression pattern in C. semilaevis when challenged with antigenic agents namely, lipopolysaccharide (LPS) or Vibrio anguillarum employing absolute quantitative real-time PCR. The real-time PCR was performed based on the standard curve generated from recombinant plasmids. No significant differences in β-actin expression were found between treated and untreated tissue samples. We thus conclude that β-actin could be used as a reliable internal reference gene for real-time PCR based quantitation of gene expression studies in various tissue samples of C. semilaevis challenged with LPS or pathogenic bacteria. [Copyright &y& Elsevier]