Abstract Background Since the late 1970s, experimental animal studies have been carried out on the possible effects of low-intensive radiofrequency fields on the blood–brain barrier (BBB), but no epidemiological study has been published to date. Objective Using serum S100B as a putative marker of BBB dysfunction we performed a descriptive cross-sectional study to investigate whether protein levels were higher among frequent than non-frequent users of mobile and cordless desktop phones. Method One thousand subjects, 500 of each sex aged 18–65 years, were randomly recruited using the population registry. Data on wireless phone use were assessed by a postal questionnaire and blood samples were analyzed for S100B. Results The response rate was 31.4%. The results from logistic and linear regression analyses were statistically insignificant, with one exception: the linear regression analysis of latency for UMTS use, which after stratifying on gender remained significant only for men ( p = 0.01; n = 31). A low p-value (0.052) was obtained for use of cordless phone ( n = 98) prior to giving the blood samples indicating a weak negative association. Total use of mobile and cordless phones over time yielded odds ratio (OR) 0.8 and 95% confidence interval (CI) 0.3–2.0 and use on the same day as giving blood yielded OR = 1.1, CI = 0.4–2.8. Conclusions This study failed to show that long- or short-term use of wireless telephones was associated with elevated levels of serum S100B as a marker of BBB integrity. The finding regarding latency of UMTS use may be interesting but it is based on small numbers. Generally, S100B levels were low and to determine whether this association – if causal – is clinically relevant, larger studies with sufficient follow-up are needed.