Comparative genomics has provided us with a new handle on the interpretation of protein sequences. The sequencing of numerous fungal genomes has offered the possibility to compare genomes over a degree of evolutionary divergence. More importantly, the large number of sequenced genomes provides for a statistical analysis of orthologues, which allows analysis of protein size in addition to primary sequence. Like protein structure, size is expected to be conserved between orthologues. As significant deviations in size are indicative of altered protein domain structure, we propose size analysis as a supplementary tool for interspecies sequence comparison. Herein, we summarize a comparative analysis of the yeast HOG pathway and highlight the use of protein size analysis in identification and comparison of orthologues, and for evaluation of sequencing and annotation. Such comparisons were used to define differences in the signalling pathway architecture between species, such as absent components or components with altered domain structure. Furthermore, they provide a powerful complement to functional protein analysis, such as discovery of novel, and evaluation of characterised, functional domains, as well as evaluation of potential sites for posttranslational modification. The fungal genome sequences provide a unique resource that should facilitate the functional analysis of any fungal proteins.