Using an ATP analog that is a specific substrate for an analog-specific allele of v-Src, we identified several novel cytoskeletal substrates that control actin assembly processes. A screen for less abundant v-Src substrates revealed the scaffolding protein Dok-1 as a direct substrate of v-Src. Further studies suggest that v-Src phosphorylation sites on Dok-1 are critical for its binding to RasGAP and Csk, negative regulators of Src signaling. This results in the downregulation of growth-promoting signals of the Src family kinases and the Ras pathway. Identification of the direct substrates of v-Src leads to a model for the precise order of assembly of a retrograde signaling pathway in v-Src-transformed cells and has provided new insight into the balance between those signals that promote cell transformation mediated by v-Src catalyzed tyrosine phosphorylation and those that inhibit it.