The mechanism that leads to the inverse relationship between heightened cellular proliferation and the cessation of elastic fibers production, observed during formation of the arterial occlusions and dermal scars, is not fully understood. Because the retinoblastoma protein (Rb), responsible for cell cycle initiation, has also been implicated in insulin-like growth factor-I-mediated signaling stimulating elastin gene activation, we explored whether differential phosphorylation of Rb by various cyclin·cyclin-dependent kinase complexes would be responsible for promoting either elastogenic or pro-proliferative signals. We first tested cultures of dermal fibroblasts derived from Costello syndrome patients, in which heightened proliferation driven by mutated oncogenic H-Ras coincides with inhibition of elastogenesis. We found that Costello syndrome fibroblasts display elevated level of Rb phosphorylation on serine 780 (Ser(P)-780-Rb) and that pharmacological inhibition of Ras with radicicol, Mek/Erk with PD98059, or cyclin-dependent kinase 4 with PD0332991 not only leads to down-regulation of Ser(P)-780-Rb levels but also enhances Rb phosphorylation on threonine-821 (Thr(P)-821-Rb), which coincides with the recovery of elastin production. Then we demonstrated that treatment of normal skin fibroblasts with the pro-proliferative PDGF BB also up-regulates Ser(P)-780-Rb levels, but treatment with the pro-elastogenic insulin-like growth factor-I activates cyclinE-cdk2 complex to phosphorylate Rb on Thr-821. Importantly, we have established that elevation of Thr(P)-821-Rb promotes Rb binding to the Sp1 transcription factor and that successive binding of the Rb-Sp1 complex to the retinoblastoma control element within the elastin gene promoter stimulates tropoelastin transcription. In summary, we provide novel insight into the role of Rb in mediating the inverse relationship between elastogenesis and cellular proliferation.