We investigated the effect of the functional insertion/ deletion (I/D) polymorphism in the angiotensin- converting enzyme (ACE) gene on the response to interferon-β (IFN-β) therapy in Croatian and Slovenian patients with multiple sclerosis (MS). A total of 275 IFN-β treated MS patients [162 responders (Rs) and 113 nonresponders (NRs)] were genotyped by PCR. The ACE I/D genotype distribution and allele frequencies did not differ between female Rs and NRs. However, male NRs tended to have a greater prevalence of the DD genotype (P=0.073 ; odds ratio: 2.64 ; 95% confidence interval: 0.91–7.60) and a significantly higher frequency of the D allele (P=0.022 ; odds ratio: 2.43 ; 95% confidence interval: 1.13–5.20) than male Rs. Multiple forward stepwise regression analysis indicated that the negative response to IFN-β therapy was associated with the ACE-DD genotype in men ( β=0.371 ; multiple R2 change: 0.132 ; P=0.009) and a higher pretreatment relapse rate in both men ( β=−0.438 ; multiple R2 change: 0.135 ; P=0.015) and women ( β=−0.208 ; multiple R2 change: 0.042 ; P=0.034). The ACE I/D polymorphism and pretreatment relapse rate accounted for ∼26.7% of the IFN-β response variability among the men in the sample. Further studies of a larger number of MS patients from different populations are necessary to evaluate these preliminary findings.