Relaxin is a naturally occurring regulator of collagen turnover. In this study, we determined the role of endogenous relaxin (RLX) in the pathogenesis of primary tubulointerstitial fibrosis after unilateral ureteric obstruction (UUO). Four-six week old relaxin gene-knockout (RLX-/-) and age-matched wild-type (RLX+/+) mice, with equivalent baseline collagen levels, were subjected to UUO. Obstructed and contralateral kidneys were collected at days-0, -3 and -10, post-surgery and analyzed for changes in inflammatory and fibrosis-related markers. UUO was associated with a progressive increase in fibrosis in all obstructed, but not contralateral kidneys. The increase in total collagen (hydroxyproline analysis) was associated with more -smooth muscle actin (-SMA) staining (myofibroblasts) and interstitial collagen sub-types (SDS-PAGE; types I, III and V), while gelatin zymography demonstrated increased expression of MMP-2 after surgery. By day-10 post-UUO there was a 5-fold decrease in RLX mRNA expression (quantitative RT-PCR) in RLX+/+ animals. Total collagen and -SMA expression were significantly greater in the obstructed kidneys of RLX-/- mice 3-days post-UUO (both P < 0.05 vs. RLX+/+ D3 post-UUO), but comparable to that in RLX+/+ animals 10-days post-UUO. Administration of recombinant H2 relaxin to RLX-/- mice, 4-days before UUO ameliorated the increase in collagen and -SMA expression (both P < 0.05 vs. untreated RLX-/- mice) by day-3 post-UUO. Expression of monocyte chemoattractant protein-1 and macrophage infiltration (inflammation) in addition to that of MMPs was unaffected by genotype post-UUO. These combined data demonstrate that endogenous RLX acts as a modulating factor in tubulointerstitial fibrosis, a hallmark of progressive renal disease. This is likely to be via direct effects on renal myofibroblast function.