There is still debate over the optimal dosage, frequency and route of administration of interferon (IFN) beta in multiple sclerosis (MS). A prospective, non-randomized, comparative study was performed to evaluate differences in magnetic resonance imaging and clinical outcomes of two IFN beta-1a preparations (30mcg intramuscular [im] once-weekly [qw], AVO; and 22 mcg subcutaneous [sc] three-times-weekly [tiw]; R22). Relapsing-remitting MS patients on one of the two IFN preparations (AVO, n=47; R22, n=48) were assessed at baseline and after 6 months of further treatment. There were no significant differences between the two groups at baseline. Both groups showed significantly reduced relapse rates (F=19.5; p<0.001) from baseline (0.6) to 6-month assessment (0.2; p<0.001). Univariate analysis showed a significant difference in favour of R22 on T2 lesion volume (F=14.4; p<0.001) and T1 black hole lesion load (F=8.5; p=0.004), the latter showing a significant increase in the AVO group (p<0.001). The incidence of patients with new T1 black holes was also higher for AVO than R22 (23.5% vs 8.3%; p=0.025). These results from patients receiving AVO or R22 in normal clinical practice are in line with randomized clinical studies that show the benefits of high-dose, high-frequency administration of IFN beta-1a in MS therapy.