Serum alanine transaminase (ALT) levels are used to select hepatitis C virus (HCV)-infected patients for treatment and liver biopsy. However, the natural history of these measurements is poorly understood. To examine the hypothesis that ALT levels vary over time in HCV-infected patients, serial serum ALT levels were prospectively measured in a cohort of 1,235 persons with a history of prior illicit drug use. Over 25 months of follow-up, there was a median of four evaluations per patient. ALT values were higher in 1,164 (94%) HCV-infected individuals than in 71 (6%) HCV-uninfected individuals. The remainder of the analysis focused on these HCV-infected individuals, 647 (62%) of whom had normal ALT values at their initial visit. However, 323 (49%) of these had at least one elevated ALT over the next 25 months. Of the 395 patients whose ALT was initially abnormal, 332 (84%) had at least one normal value over the next 25 months. Overall, among individuals with four or more visits, ALT values were persistently normal in 42%, persistently elevated in 15%, and intermittently elevated in 43%. Because serum ALT levels have high visit-to-visit variability, single assessments should not be used to manage HCV-infected individuals. Further investigation is needed to ascertain the correlation of serial ALT trends with important disease outcomes.