This study describes a new computational approach for studying the evolutionary rewiring of transcriptional regulatory networks by changes in the targets of transcription factors. By applying the approach to the study of 88 transcription factor target binding motifs in 23 species of ascomycete fungi, the authors found that although the biological functions of regulatory networks are conserved, there is considerable plasticity in transcription factor target genes. This suggests a model in which there is selection to maintain the overall function of transcription factors but plasticity with respect to specific targets.