Complete nucleotide sequences have been determined for atp6 from Suillus luteus and cox3 from Suillus sinuspaulianus (Boletales, Hymenomycetes, Basidiomycota), which code for ATPase subunit 6 and cytochrome oxidase subunit 3, respectively. These sequences were used to design PCR primers for the amplification of partial atp6 and cox3 sequences from other members of the Boletales and outgroup taxa. In atp6 and cox3 from Russula rosacea, one of the outgroup taxa, we observed a number of in-frame TGA trp codons, which imply a Neurospora crassa-type mitochondrial code in R. rosacea and possibly in basidiomycetes in general. Interestingly, however, most basidiomycetes other than R. rosacea appear to strongly prefer the TGG trp codon, which is unusual, given the strong A Ψ T bias in fungal mitochondrial genomes. Pairwise comparisons were performed between atp6 sequences from increasingly divergent fungal lineages, and results show that all three codon positions become saturated in substitutions after an estimated divergence time of approx 300 Ma. This means that atp6 is likely to provide phylogenetic resolution within fungal classes but not at higher taxonomic levels. Also, because of the strong A Ψ T bias in fungal mitochondrial genomes, A/T transversions were found to be more common than any other type of substitution, resulting in transversions being about two to three times more common in most pairwise sequence comparisons. Finally, atp6 sequences were used to infer phylogenetic relationships between 27 taxa from the Boletales and 4 outgroup taxa. Analyses were performed (i) on nucleotide sequence data using parsimony (successive approximation) as well as maximum likelihood methods and (ii) on deduced amino acid sequences using distance methods based on empirical substitution probabilities. Results from the various analyses are largely concordant with each other as well as with prior analyses of partial mitochondrial large-subunit rDNA (mtLSU rDNA). Analysis of the combined atp6 and mtLSU rDNA sequences results in increased bootstrap support for several key branches. Relationships that have been resolved for the first time in the current analysis are discussed. 1999 Academic Press