The inoperculate euascomycetes are filamentous fungi that form saprobic, parasitic, and symbiotic associations with a wide variety of animals, plants, cyanobacteria, and other fungi. The higher-level relationships of this economically important group have been unsettled for over 100 years. A data set of 55 species was assembled including sequence data from nuclear and mitochondrial small and large subunit rDNAs for each taxon; 83 new sequences were obtained for this study. Parsimony and Bayesian analyses were performed using the four-region data set and all 14 possible subpartitions of the data. The mitochondrial LSU rDNA was used for the first time in a higher-level phylogenetic study of ascomycetes and its use in concatenated analyses is supported. The classes that were recognized in Leotiomyceta (=inoperculate euascomycetes) in a classification by Eriksson and Winka [Myconet 1 (1997) 1] are strongly supported as monophyletic. The following classes formed strongly supported sister-groups: Arthoniomycetes and Dothideomycetes, Chaetothyriomycetes and Eurotiomycetes, and Leotiomycetes and Sordariomycetes. Nevertheless, the backbone of the euascomycete phylogeny remains poorly resolved. Bayesian posterior probabilities were always higher than maximum parsimony bootstrap values, but converged with an increase in gene partitions analyzed in concatenated analyses. Comparison of five recent higher-level phylogenetic studies in ascomycetes demonstrates a high degree of uncertainty in the relationships between classes.