Abstract: ABSTRACT: BACKGROUND: Microsporidia, parasitic fungi-related eukaryotes infecting many cell types in a wide range of animals (including humans), represent a serious health threat in immunocompromised patients. The 2.9 Mb genome of the microsporidium Encephalitozoon cuniculi is the smallest known of any eukaryote. Eukaryotic protein kinases are a large superfamily of enzymes with crucial roles in most cellular processes, and therefore represent potential drug targets. We report here an exhaustive analysis of the E. cuniculi genomic database aimed at identifying and classifying all protein kinases of this organism with reference to the kinomes of two highly-divergent yeast species, Saccharomyces cerevisiae and Schizosaccharomyces pombe. RESULTS: A database search with a multi-level protein kinase family hidden Markov model library led to the identification of 29 conventional protein kinase sequences in the E. cuniculi genome, as well as 3 genes encoding atypical protein kinases. The microsporidian kinome presents striking differences from those of other eukaryotes, and this minimal kinome underscores the importance of conserved protein kinases involved in essential cellular processes. ~30% of its kinases are predicted to regulate cell cycle progression while another ~28% have no identifiable homologues in model eukaryotes and are likely to reflect parasitic adaptations. E. cuniculi lacks MAP kinase cascades and almost all protein kinases that are involved in stress responses, ion homeostasis and nutrient signalling in the model fungi S. cerevisiae and S. pombe, including AMP activated protein kinase (Snf1), previously thought to be ubiquitous in eukaryotes. A detailed database search and phylogenetic analysis of the kinomes of the two model fungi showed that the degree of homology between their kinomes of ~85% is much higher than that previously reported. CONCLUSION: The E. cuniculi kinome is by far the smallest eukaryotic kinome characterised to date. The difficulty in assigning clear homology relationships for nine out of the twenty nine microsporidian conventional protein kinases despite its compact genome reflects the phylogenetic distance between microsporidia and other eukaryotes. Indeed, the E. cuniculi genome presents a high proportion of genes in which evolution has been accelerated by up to four-fold. There are no orthologues of the protein kinases which constitute MAP kinase pathways and many other protein kinases with roles in nutrient signalling are absent from the E. cuniculi kinome. However, orthologous kinases can nonetheless be identified that correspond to members of the yeast kinomes with roles in some of the most fundamental cellular processes. For example, E. cuniculi has clear orthologues of virtually all the major conserved protein kinases that regulate the core cell cycle machinery (Aurora, Polo, DDK, CDK and Chk1). A comprehensive comparison of the homology relationships between the budding and fission yeast kinomes indicates that, despite an estimated 800 million years of independent evolution, the two model fungi share ~85% of its protein kinases. This will facilitate the annotation of many of the as yet uncharacterised fission yeast kinases, and also those of novel fungal genomes.