Mdr2 (Abcb4)-/- mice develop hepatic lesions resembling primary sclerosing cholangitis. Our aim was to characterize the evolution of fibrosis in Mdr2-/- mice. Mdr2-/-mice and their wild-type littermates were sacrificed at 2, 4 and 8 weeks after birth. Hepatic collagen was determined biochemically. Fibrosis related transcript levels were quantified from livers by real-time RT-PCR, and MMP activities determined by substrate assays. Liver histology was assessed by connective tissue staining and immunohistochemistry for alpha-smooth muscle actin (alpha-SMA). Mdr2-/- mice demonstrated a time-dependent increase of relative and total hepatic collagen (fivefold at 8 weeks, compared to wildtype controls), and maximal alpha-SMA immunoreactivity at 4 weeks. Compared to wildtype controls profibrogenic mRNA levels for procollagen alpha1(I), TGFbeta1, TGFbeta2, MMP-2 and -13, TIMP-1, PDGFbeta receptor, and PAI-1 were upregulated up to 27-fold. Most transcripts peaked at 4 weeks, but procollagen alpha1(I) mRNA increased steadily, TIMP-1 mRNA was constantly elevated (20-fold), MMP-13 mRNA was suppressed and interstitial collagenase and gelatinase activities were downregulated. Mdr2-/- mice spontaneously progress to severe biliary fibrosis. This is due to a characteristic temporal pattern of upregulated profibrogenic and downregulated fibrolytic genes and activities. These mice are an attractive model to test potential antifibrotics for the treatment of (biliary) liver fibrosis.