Renal myofibroblasts play a crucial role in the accumulation of excess extracellular matrix during renal fibrosis. Both transforming growth factor-beta1 (TGFbeta1) and connective tissue growth factor (CTGF) are important profibrotic growth factors, which interact in the pathogenesis of fibrosis. In this study, we demonstrate that CTGF alone has no influence on myofibroblast transformation and fibronectin secretion in kidney interstitial fibroblasts, whereas incubation of CTGF in combination with TGFbeta1 enhanced TGFbeta1 responses, including myofibroblast activation, de novo expression of alpha-SMA, and extracellular accumulation of fibronectin. CTGF induced tryrosine phosphorylation of the cytoplasmic domain of the low-density lipoprotein receptor-associated protein (LRP) in fibroblasts, and the LRP-antagonist, receptor-associated protein (RAP) inhibited CTGF-induced tryrosine phosphorylation of LRP. Inhibition of LRP signaling reduced CTGF-mediated synergistic induction of alpha-SMA protein. Furthermore, the potentiating action of CTGF was neither dependent on modulation of TGFbeta1-induced Smad2 phosphorylation and its association with Smad4, nor did it result from nuclear accumulation of activated Smad2. When TGFbeta1-pretreated fibroblasts were incubated with CTGF, activation of ERK1/2 MAPK signaling was observed. Inhibition of ERK activation by the MEK1 inhibitor PD98059 was associated with a reduction of CTGF-promoted alpha-SMA protein expression. Our in vitro studies provide evidence that CTGF potentiates TGFbeta1-mediated myofibroblast differentiation and activates differentiated myofibroblasts.