The E1 and E2/NS1 genes, encoding the putative hepatitis C virus envelope proteins, show a high rate of sequence variations. We analyzed the degree and distribution of sequence heterogeneity in serum samples from hepatitis C virus-infected subjects. The mutations in the E1 region were mainly type-specific and the rate of variability was apparently not linked to the clinical phase of the infection. The sequence evolution of the E1 region during interferon treatment was low, regardless of the response to therapy. In contrast, an increased degree of variation, apparently related to the stage of viral replication, was present in E2 region derived from patients undergoing interferon treatment. These results are consistent with the hypothesis that the E2 protein represents a major target of the immune response.