The extent of co-expression of the components of a comprehensive set of 244 multi-subunit protein complexes over large DNA microarray data sets was investigated in S. cerevisiae, A. thaliana, C. elegans, D. melanogaster and H. sapiens. Co-expression amongst the components of protein complexes is substantial and occurs in all the species studied, reflecting the importance of regulation at the level of the transcript. Complexes that are conserved in all species, and tend to be associated with very fundamental biological processes, are significantly more likely to have co-expressed components, indicating the fundamental and ancient evolutionary nature of transcriptional regulation. Less conserved complexes are more likely to have evolved alternative regulatory mechanisms. Within the conserved complexes there is evidence that regulatory mechanisms (transcriptional or other) are evolutionarily conserved, but there are exceptions. The fact that complexes that share subunits are more likely to share co-expression is further evidence of evolutionary conservation of regulatory mechanisms. This extensive study contributes to the fundamental biological understanding of important protein complexes by enabling evolutionary insights into their regulation through a new multi-genome perspective. In addition, it is relevant to developers and users of gene function prediction methods that use gene co-expression to indicate potential functional linkage.