We analyzed the effect of beta-interferon (beta-IFN) treatment over the active replication of JC virus (JCV) through the evaluation of JCV DNA prevalence and viral load in peripheral blood mononuclear cells (PBMCs) and serum samples, and mRNA prevalence and viral load, in relapsing-remitting multiple sclerosis (RRMS) patients. DNA extracted from PBMCs and serum, and mRNA extracted from PBMCs were analyzed in 146 RRMS patients (73 treated with beta-IFN, and 73 untreated patients), and 73 matched healthy blood donors for the presence of JCV genomes by quantitative real-time PCR assay. We found the same DNA prevalence in PBMC samples in RRMS patients treated with beta-IFN and in untreated ones: 6.8% (5/73). When we analyzed the viral active replication in both groups through the analysis of DNA prevalence in serum samples and the mRNA extracted from PBMCs, we did not find any positive sample. Regarding the viral load of those positive samples, we did not find any statistical significant difference between treated and untreated RRMS patients: 28.6 +/- 7.2 and 32.3 +/- 8.4 copies/microg of DNA, respectively. These results lead us to conclude that beta-IFN treatment in monotherapy has not any effect on JCV active replication.