The cardiotonic steroid, marinobufagenin (MBG) has been implicated in the pathogenesis of experimental uremic cardiomyopathy which is characterized by progressive cardiac fibrosis. We examined whether the transcription factor, Fli-1 might be involved in this process. Fli-1 knockdown demonstrated greater cardiac collagen-1 expression and fibrosis compared to wild type mice; both developed increased cardiac collagen expression and fibrosis following 5/6th nephrectomy. There was a strong inverse relationship between the expressions of Fli-1 and procollagen in primary culture of rat cardiac and human dermal fibroblasts as well as a cell line derived from renal fibroblasts, and MBG induced decreases in nuclear Fli-1 as well as increases in procollagen-1 expression in these cells. Transfection of a Fli-1 expression vector prevented increased procollagen-1 expression from MBG. MBG exposure induced a rapid translocation of the delta isoform of protein kinase C (PKC) to the nucleus. This translocation was prevented by pharmacological inhibition of phospholipase C, and MBG induced increases in procollagen-1 expression were prevented with a PKC but not a PKC specific inhibitor. Finally, immunoprecipitation studies strongly suggest that MBG induced phosphorylation of Fli-1. We feel these data support a causal relationship between MBG induced translocation of PKCwhich result in phosphorylation of as well as decreases in nuclear Fli-1 expression which, in turn, leads to increases in collagen production. Should these findings be confirmed, we speculate that this pathway may represent a therapeutic target for uremic cardiomyopathy as well as other conditions associated with excessive fibrosis.