Elevated levels of homocysteine have been observed in Parkinson's disease (PD) patients treated with levodopa. However, it is not studied if duration of PD or PD per se is associated with hyperhomocysteinemia. In the present study, the levels of homocysteine in 99 levodopa-treated PD patients, 15 untreated PD patients and 100 controls were examined. We focused on the influence of levodopa dose, duration of therapy and disease as well as genetic (C677T methylenetetrahydrofolate reductase (MTHFR) polymorphism) and environmental factors. We found that levodopa-treated PD patients had elevated homocysteine plasma levels as compared to controls (p < 0.05), but the levels did not depend on levodopa doses. Another factor influencing homocysteine level was the duration of PD (p < 0.001). The frequency of allele C677T of MTHFR gene did not differ between PD and controls. In conclusion, hyperhomocysteinemia is associated with the duration of PD and levodopa treatment and possibly also with PD per se.