It is assumed that testosterone is an important regulator of gender-related differences in ventricular repolarization. Therefore, our aim was to study whether serum levels of testosterone are associated with QTc, QT and RR interval variation. Setting: two independent population-based cohort studies. Participants: 445 male participants (≥ 55 years) from the Rotterdam study cohort and 1,428 male participants from the study of health in Pomerania (SHIP) with an electrocardiogram who were randomly sampled for assessment of serum testosterone at baseline, after exclusion of participants with testosterone altering drugs, QTc prolonging drugs or dig(it)oxin, left ventricular hypertrophy and left and right bundle branch block. Endpoints: length of the QTc, QT and RR intervals. Analysis: linear regression model, adjusted for the two individual studies and a pooled analysis of both studies. The pooled analysis of the Rotterdam study and SHIP showed that the QTc interval gradually decreased among the tertiles (P value for trend 0.024). The third tertile of serum testosterone was associated with a lower QTc interval compared to the first tertile [-3.4 ms (-6.5; -0.3)]. However, the third tertile of serum testosterone was not associated with a lower QT interval compared to the first tertile [-0.7 ms (-3.1; 1.8)]. The RR interval gradually increased among the tertiles (P value for trend 0.002) and the third tertile of serum testosterone showed an increased RR interval compared to the first tertile [33.5 ms (12.2; 54.8)]. In the pooled analysis of two population-based studies, serum testosterone levels were not associated with the QT interval, which could be due to a lack of power. Lower QTc intervals in men with higher serum testosterone levels could be due to the association of serum testosterone with prolongation of the RR interval.