Despite substantial benefits of HAART treatment of HIV-infected patients, cumulative long-term toxicity, including drug-induced hepatotoxicity, has emerged as an important complication. Thus, to examine the prevalence and risk of developing severe hepatic injury during HAART, we conducted a retrospective study in a cohort of 364 HIV-infected patients treated with HAART between January 1998 and May 2006, for whom data on alanine aminotransferase activity were available both before and during HAART. HCV co-infection was recorded in 35.4% of the series, but was found not to influence either the efficacy of HAART or survival (P>0.05). Severe hepatotoxicity occurred in a total of 24 patients (6.6%). Multivariate logistic regression defined HCV co-infection (OR 16.6, 95% CI 3.8-46.0, P<0.0001), and the use of SQV/RTV and d4T (OR 3.1, 95% CI 1.2-8.16, P=0.02, and OR 7.1, 95% CI 1.0-54.5, P=0.05, respectively) as independent risk factors for aggravation of hepatitis. In addition, there was a significant increase in the probability of developing liver damage over years of treatment (Log rank, P<0.01). Conversely, the probability of developing hepatotoxicity was not associated with an increase in the CD4 cell count to values greater than 350/μL (Log rank, P=0.59). In conclusion, in the setting of chronic viral hepatitis, hepatotoxicity during HAART may be attributed to the cumulative toxicity of drugs that induce mitochondrial toxicity, along with particular PIs and/or NNRTIs. Furthermore, our data suggest prudent use of D-drugs, still common in resource-limited countries, in HCV co-infected patients.