The importance of elevated homocysteine (Hcy) as a risk marker for cardiovascular disease is continuously under debate. Lifestyle factors may increase the total Hcy (tHcy) level of the plasma, but there are no consistent findings relating to Hcy, physical activity, and cardiorespiratory fitness. Cross-sectional measurement from an ongoing follow-up study was performed on 77 former male athletes and 33 sedentary controls (age range 35-62 years). Lifestyle parameters (current physical activity patterns, smoking, etc.), anthropometric and blood pressure data, and data about tHcy, reduced, and oxidized glutathione (GSH, GSSG, respectively) in blood, lipoproteins, and maximal oxygen consumption (VO(2max)) were collected. Our study results showed that the subgroup of physically active ex-athletes (n=52) had a significantly lower tHcy level and glutathione redox ratio (GSSG:GSH) in comparison with the subgroup of sedentary ex-athletes (n=25). tHcy level was inversely related to cardiorespiratory fitness (VO(2max)/kg). Dietary and smoking habits were not significantly associated with the tHcy level in our study group. In conclusion, the research findings indicate that both current physical activity and cardiorespiratory fitness are significantly inversely associated with an elevated homocysteine level in middle-aged former athletes.