Molecular systematics involves the description of the regulatory networks formed by the interconnections between active transcription factors and their target expressed genes. Here, we have determined the activities of 200 different transcription factors in six mouse tissues using an advanced mouse oligonucleotide array-based transcription factor assay (MOUSE OATFA). The transcription factor signatures from MOUSE OATFA were combined with public mRNA expression profiles to construct experimental transcriptional regulatory networks in each tissue. SRF-centered regulatory networks constructed for lung and skeletal muscle with OATFA data were confirmed by ChIP assays, and revealed examples of novel networks of expressed genes coregulated by sets of transcription factors. The combination of MOUSE OATFA with bioinformatics analysis of expressed genes provides a new paradigm for the comprehensive prediction of the transcriptional systems and their regulatory pathways in mouse.