Fibres of cotton (Gossypium hirsutum L.) are single elongated epidermal cells that start to develop on the outer surface of cotton ovules on the day of anthesis. Little is known about the control of fibre initiation and development. As a first step towards discovering important genes involved in fibre initiation and development using a genomics approach, we report technical advances aimed at reducing redundancy and increasing coverage for anonymous cDNA microarrays in this study. Cotton ovule cDNA libraries (both normalised and un-normalised) from around the time of fibre initial formation have been prepared and partially characterised by sequencing. Re-association-based normalisation partially reduced library redundancy and increased representation of novel sequences. However, another library generated from in vitro cultured cotton ovules treated with the protein synthesis inhibitor, cycloheximide, showed a significantly altered gene representation including a greater proportion of protein phosphorylation genes, transport genes and transcription factors and a much reduced proportion of protein synthesis genes than were identified in the conventional types of libraries. Over 10,000 expressed sequence tag (EST) clones randomly selected from the three libraries were printed on microarray slides and used to assess gene expression in tissue cultured ovules with and without cycloheximide treatment. The microarray results showed that cycloheximide had a dramatic effect in modifying the pattern of the gene expression in cultured ovules, affecting the same types of genes identified in the preliminary analysis on relative EST abundance in the different ovule cDNA libraries. Cycloheximide clearly provided a simple and useful method for enriching novel gene sequences for genomic studies.