This study examined the role of agonist-induced Rho kinase (ROCK) involvement in the morphological outcome of pulmonary-derived fibroblasts. Normal human lung fibroblasts (NHLF) spontaneously differentiate into network-like structures in a two-dimensional growth factor reduced Matrigel matrix-based assay. Sphingosine 1-phosphate (SPP), a bioactive phospholipid that regulates angiogenesis, inhibited fibroblast morphogenesis in a dose-dependent manner, virtually eliminating the presence of multi-cellular structures at 500 nM. Pretreatment with the Rho kinase-specific inhibitor, H1152, eradicated the high dose SPP-induced inhibition. Similarly, NHLFs transfected with Rho kinase siRNA prevented SPP-induced inhibition of the fibroblast morphogenesis. Alternatively, transforming growth factor-beta1 (TGF-beta1), a cytokine recognized as a key mediator of wound healing, terminally differentiates NHLF into myofibroblasts as evidenced by the expression of the smooth muscle cell isoform of alpha-actin (alpha-SMA). H1152 suppressed TGF-beta1-induced alpha-SMA expression in a dose-dependent manner. Similarly, treatment with Rho kinase siRNA reduced alpha-SMA expression by greater than 50%. SPP treatment had no effect on TGF-beta1-induced transformation into myofibroblasts, and TGF-beta1 treatment did not alter fibroblast morphogenesis. This study suggests a dual regulatory role for Rho kinase in cellular regulation of fibroblasts in which SPP-induced Rho kinase activation via a G-protein coupled receptor suppresses fibroblast morphogenesis while TGF-beta1-induced Rho kinase activation through a serine/threonine kinase receptor culminates in transformation into myofibroblasts.