Chronic allograft nephropathy (CAN) is characterized by progressive renal functional loss and histologic abnormalities of one or more tissue compartments. In this study, correlations between histologic abnormalities and graft function [glomerular filtration rate (GFR, measured by iothalamate clearance), serum creatinine (SCr) and urinary protein (UPr)] were investigated using biopsies from 49 patients with newly diagnosed CAN. Extent of tubulointerstitial fibrosis (%TIF), as assessed by a semi-quantitative score, correlated significantly with GFR, SCr and UPr. The close correlation between %TIF and GFR suggested that quantitative measurement of %TIF may predict functional consequences of CAN. Calculation of %TIF by computerized digital analysis was performed using four strategies: (a) quantitation of blue material in Masson's trichrome (MT)-stained sections, (b) quantitation of red material in Sirius Red-stained sections (SR-nonpolarized), (c) quantitation of birefringent material in Sirius Red stained-sections examined under polarized light (SR-polarized) and (d) quantification of brown material in sections stained by immunoperoxidase for alpha-smooth muscle actin. Only the SR-nonpolarized score correlated significantly with GFR at the time of biopsy-diagnosis of CAN. We conclude that digital analysis strategies demonstrate variable accuracy in quantifying %TIF. Validation of the SR-nonpolarized strategy against histologic scoring and GFR supports the application of this technique to longitudinal studies of CAN.