Beta2-microglobulin (beta2-MG) is a pharmacodynamic marker of interferon-beta activity in multiple sclerosis (MS). Its role in the natural course of the disease is not fully known. We analyzed the spontaneous fluctuation of beta2-MG in free-treatment MS patients during a short-time course to quantify beta2-MG as a marker of disease activity/progression. Thirty MS patients were clinically assessed and imaged monthly over a 3-month period. Sera were collected concomitantly for the evaluation of beta2-MG, by means of an enzyme-linked immunosorbent assay. Sera from 20 healthy individuals (HI) were drawn and used as controls. The Mann-Whitney test was used when appropriate and time effect on radiological and biological measures was assessed by means of the random effect models. Eight (26.7%) patients experienced a clinical relapse but three (10%) required steroid treatment. A reduction in the contrast-enhancing lesion load (P = 0.02) and a trend (P = 0.07) toward a decrease in brain parenchyma fraction were observed. Baseline levels of beta2-MG were similar in patients and HI. Patients' beta2-MG values increased over the 3-month time period (P = 0.05) but did not exceed those detected in HI at any time point. These results failed to demonstrate the validity of beta2-MG as a surrogate marker of disease in MS.