Changes in gene expression in liver of medaka in response to 4-nonylphenol (4-NP) were investigated with a microarray consisting of 3.4 K medaka-specific cDNA probes. Upon exposure to 4-NP at two different concentrations (20 and 100 microg/L) for 24 h, we found 44 different genes that were up- or downregulated with statistically significant changes. Digestive enzyme genes were downregulated but vitellogenin, choriogenin and novel biomarker genes were upregulated. Additionally an analysis of the changes of gene expression with respect to time was conducted at 100 microg/L of 4-NP with samples at 0, 24, 48 and 96 h. The expression of 75 genes was altered and these were categorized into four different clusters. The validity of the results was checked by semi-quantitative PCR with 12 representative genes and this confirmed the microarray data. Therefore, we suggest that this medaka 3.4 K chip would be useful in generating gene expression profiles for studies of ecotoxicogenomics in medaka.