The immune system is regulated at many levels by microRNA action, including differentiation and proliferation of myeloid lineages, and cellular responses to proinflammatory stimuli. Several recent studies have reported the presence of microRNAs within human neutrophils, yet their role in defining neutrophil functions has not been reported. Mature microRNAs are 20–25 nucleotides in length and are produced from a primary transcript, usually several kilobases long, which is cleaved in the nucleus by Drosha and its cofactor DGCR8 to leave a premiRNA of about 70bp. Pre-miRNA is then transported into the cytosol by exportin 5 followed by cleavage by Dicer to leave mature microRNA. There are currently over 850 human microRNAs and they are thought to regulate up to 30% of the human genome. Due to incomplete basepairing with the target sequence, a single microRNA can target many hundreds of genes, and a single gene can be regulated by multiple microRNAs, allowing fine tuning of gene transcription. Key determinants of neutrophil functional longevity, such as Mcl-1, are known to be regulated by microRNAs in other cell types. We therefore examined changes in microRNA expression over time and upon treatment with GMCSF in human neutrophils, revealing for the first time the co-ordinated dynamic regulation of microRNAs, and hence a range of target genes with roles in regulation of neutrophil function. This work begins to identify how neutrophils use microRNAs as one of the tools available to limit the proinflammatory potential of these essential, but potentially harmful host defence cells. The microRNAs identified were miR-16, which has been previously described as suitable for normalisation in a RT-PCR study of human breast cancer tissue and miR-720. Fold change was then simply calculated through the subtrAG-013736 action of one value from another. Where direct comparisons were performed, each sample was within the same chip. Elimination of monocyte contamination is critical in these experiments, since monocytes have been shown to modulate neutrophil responses to inflammatory stimuli and may theoretically contribute disproportionately to the detectable microRNA population. In contrast, eosinophil contamination has little functional effect on neutrophils, and is unlikely to contribute significantly to the microRNA profiles seen. However, a detailed analysis of eosinophil miRNAs has not been published, and the possibility that some of the findings may be due to eosinophil contamination cannot be completely excluded. Following preparation of sufficiently high quality total RNA, samples were labelled, loaded onto the array slides and read following the manufacturer’s instructions. The data were extracted using Feature Extraction V10.7 and data were transferred into Microsoft Excel for analysis. In total, 146 out of the 851 microRNAs were found to be present in at least 4 out of 5 donors, as indicated by the Feature Extraction software .