By using an shRNA approach to knockdown the expression of the prostaglandin (PG)-F(2alpha) receptor (FP-R), the role of PGF(2alpha) in the process of phenotypic transformation of normal rat kidney (NRK) fibroblasts has been studied. Our data show that PGF(2alpha) up-regulates Cox-2 expression both at the mRNA and protein level, indicating that activation of FP-R in NRK fibroblasts induces a positive feedback loop in the production PGF(2alpha). Knockdown of FP-R expression fully impaired the ability of PGF(2alpha) to induce a calcium response and subsequent depolarization in NRK cells. However, these cells could still undergo phenotypic transformation when treated with a combination of EGF and retinoic acid, but in contrast to the wild-type cells, this process was not accompanied by a membrane depolarization to -20 mV. Knockdown of FP-R expression also impaired the spontaneous firing of calcium action potentials by density-arrested NRK cells. These data show that a membrane depolarization is not a prerequisite for the acquisition of a transformed phenotype. Furthermore, our data provide the first direct evidence that activity of PGF(2alpha) by putative pacemaker cells underlies the generation of calcium action potentials in NRK monolayers.