Using a nutrigenomics approach we studied the response of second-generation chickens at a transcriptional level to organically grown feed ingredients compared with conventionally grown feed ingredients. Both diets consisted of the same amounts of ingredients, the only difference was the production method. Gene expression was analysed in jejuni using whole genome chicken cDNA arrays. After analysis, forty-nine genes were found to be differentially regulated between chickens fed on the different diets, independent of their genetic background. Of these forty-nine genes, seven genes were involved in cholesterol biosynthesis. Genes involved in cholesterol biosynthesis were higher expressed in jejuni from organically fed birds. Other genes found to be regulated were involved in immunological processes, such as B-G protein (part of chicken major histocompatibility complex), chemokine ah221, and the immunoglobulin heavy chain. Using quantitative PCR the effect of genetic background on the differential expression of genes was studied. Differences in gene expression existed between animals fed different diets as well as between different chicken lines. This indicated that diet and genetic background influence the transcriptional response of the jejunum. This is the first time that significant differences in gene expression were shown between animals on diets with organically or conventionally produced ingredients.