Interstitial cells of Cajal (ICC) have important functions in regulation of motor activity in the gastrointestinal tract. In murine small intestine ICC are gathered in the regions of the myenteric plexus (ICC-MY) and the deep muscular plexus (ICC-DMP). These two classes of ICC have different physiological functions. ICC-MY are pacemaker cells and generate the slow wave electrical rhythmicity of gastrointestinal organs. ICC-DMP form synaptic connections with the varicose nerve terminals of enteric motor neurons and are involved in reception and transduction of motor neurotransmission. Gene expression underlying specific functions of ICC classes is incompletely understood. In the present study we used recently developed highly selective techniques to isolate the two functional ICC classes from enzymatically dispersed intestinal muscles by fluorescence-activated cell sorting. The transcriptome of ICC-MY and ICC-DMP was investigated using oligonucleotide microarray analysis. Differential expression of functional groups of genes defined by standard gene ontology terms was also studied. There were substantial numbers of genes expressed more abundantly in ICC than in the tunica muscularis, and we also detected marked phenotypic differences between ICC-MY and ICC-DMP. Notably, genes related to cell junction, process guidance and vesicle trafficking were up-regulated in ICC. Consistent with their specific functions, metabolic and Ca2+ transport genes were relatively up-regulated in ICC-MY while genes for signaling proteins involved in transduction of neurotransmitter functions were relatively up-regulated in ICC-DMP. Our results may lead to the identification of novel biomarkers for ICC and provide directions for further studies designed to understand ICC function in health and disease.