High level of total serum calcium within the normal range has been associated with increased cardiovascular risk. We wanted to evaluate whether the physiological active ionised form of calcium also was a risk factor for cardiovascular disease (CVD). Methods: A total of 974 participants from a health survey in 1981 in Copenhagen had ionised serum calcium and different cardiovascular risk factors measured. The participants were followed until 1999 in The National Hospital Patients Registry and The National Death Registry in Denmark regarding the diagnosis ischemic heart disease (IHD) and a broader definition of CVD. Persons with ionised serum calcium in the highest quintile were compared with persons in the lower four quintiles. Results: In a univariate analysis persons with ionised serum calcium in the highest quintile had increased risk of IHD (p=0.001) and CVD (p=0.02) compared to persons in the lower quintiles. The increased risk disappeared when gender and cardiovascular confounders were included in a Cox proportional hazards analysis (p=0.20 for IHD and p=0.50 for CVD, respectively). In a separate analysis of men and IHD the result remained insignificant (p=0.07). Conclusions: Serum ionised calcium in the upper quintile in women and men together did not seem to be a risk factor in development of IHD and CVD, but a non-significant tendency towards increased risk of IHD was observed in men with ionised calcium in the upper quintile compared to men in the lower four quintiles.