Transcriptional regulation of gene expression controls cell functions as diverse as differentiation, replication and apoptosis. The comprehensive characterization of the transcriptional mechanisms involved in these functions requires functional analysis of the approximately 2,000 transcription factors predicted in the human genome, in addition to their cognate chromosomal DNA sequences. Further increasing the functional complexity of transcriptional regulation, the activity of individual transcription factors is often modulated by post-translational modifications including phosphorylation by specific protein kinases. A novel transfection method, termed STEP (Surface Transfection and Expression Protocol), is reported here which employs microarray-based DNA transfection of adherent cells in the functional analysis of transcriptional regulation. In STEP, recombinant proteins with biological activities selected to enhance transfection are complexed with plasmid or linear expression vectors prior to spotting on microscope slides. A co-transfection assay using effector expression vectors encoding the cAMP-dependent protein kinase (PKA) as well as reporter vectors containing PKA-regulated promoters demonstrates that STEP transfection allows detection and quantitation of transcriptional regulation. This approach should be of utility in high-throughput functional genomic studies of transcriptional regulation in addition to other assays of cellular function.