Although most hepatitis C virus (HCV) infections are acquired by injection drug use, prospective data on the progression of liver fibrosis are sparse. Baseline liver biopsies were obtained (1996-1998) on a random sample of 210 out of 1667 HCV-positive injection drug users (IDUs). Subjects were followed biannually, with a second biopsy offered to those eligible. Paired biopsies were scored 0 to 6 (modified Ishak score), significant fibrosis was defined as score 3 or greater, and progression of fibrosis was defined as an increase 2 or more units or clinical evidence of end-stage liver disease. Predictive values of blood markers [FibroSURE, aspartate aminotransferase-to-platelet-ratio index (APRI) and alanine aminotransferase (ALT)] were assessed for detection of contemporaneous and future liver fibrosis. Among 119 prospectively followed IDUs, 96% were African American; 97% HCV genotype 1a/b; 27% HIV-infected, and median age was 42 years. Most (90.7%) did not have significant liver fibrosis at first biopsy. Although predictive value for detecting insignificant fibrosis at first biopsy was greater than 95% for FibroSURE, APRI, and ALT, specificities were 88.9%, 72.7%, and 72.7%, respectively. After 4.2 years median follow-up, 21% had progression of fibrosis, which was significantly associated with serum level of HCV RNA and ALT. No serological test had predictive value greater than 40% for contemporaneous or future significant fibrosis. Even initial biopsy result had only a 30.4% value for predicting future significant fibrosis. In conclusion, significant liver fibrosis and progression were detected in some, but not most, IDUs in this cohort. In this setting with low fibrosis prevalence, FibroSURE, ALT, and APRI tests predict insignificant fibrosis; however, further work is needed to find noninvasive markers of significant liver fibrosis.