We have taken an integrated approach in which expression profiling has been combined with the use of small molecule inhibitors and computational analysis of transcription factor binding sites to characterize regulatory sequences of genes that are targets of specific signaling pathways in growth factor-stimulated human cells. T98G cells were stimulated with platelet-derived growth factor (PDGF) and analyzed by DNA microarrays, which identified 74 immediate-early gene transcripts. Cells were then treated with inhibitors to identify subsets of genes that are targets of the phosphatidylinositol 3-kinase (PI3K) and MEK/ERK signaling pathways. Four groups of PDGF-induced genes were defined: independent of PI3K and MEK/ERK signaling, dependent on PI3K signaling, dependent on MEK/ERK signaling, and dependent on both pathways. The upstream regions of all genes in the four groups were scanned using TRANSFAC for putative cis-elements as compared with a background set of non-induced genes. Binding sites for 18 computationally predicted transcription factors were over-represented in the four groups of co-expressed genes compared with the background sequences (p < 0.01). Many of the cis-elements identified were conserved in orthologous mouse genes, and many of the predicted elements and their cognate transcription factors were consistent with previous experimental data. In addition, chromatin immunoprecipitation assays experimentally verified nine predicted SRF binding sites in T98G cells, including a previously unknown SRF site upstream of DUSP5. These results indicate that groups of human genes regulated by discrete intracellular signaling pathways share common cis-regulatory elements.