Mechanisms involved in the association between hyperhomocysteinemia and vascular occlusive diseases remain unclear. Homocysteine (Hcy) may disturb calcium (Ca2+) cytosolic regulation in endothelial cells, a process that can directly affect the synthesis of vasoactive substances, such as nitric oxide (NO). We have investigated the effect of acute and chronic incubation with high concentrations of Hcy (100 and 500 μmol/L) on the changes in the intracellular Ca2+ concentration ([Ca2+]i) induced by ATP, using primary cultures of human umbilical vein endothelial cells (HUVEC). The changes in [Ca2+]i, expressed as ΔFt/Fb, were measured using the microspectrofluorimetric technique with Fluo-3 as Ca2+ indicator. HUVEC acutely exposed to Hcy did not produce significant effects on any of the parameters studied. However, chronic exposition (24 h) caused a significant decrease in the speed of store-mediated Ca2+ entry, expressed as (ΔFt/Fb)/t (s−1). Exposure of HUVEC to 100 and 500 µmol/L Hcy gave significantly lower values (0.019 ± 0.002 s−1, n = 5 and 0.021 ± 0.004 s−1, n = 6, respectively) compared to the controls (0.046 ± 0.004 s−1, n = 8, P < 0.003). This was detected only when the sustained phase of the ATP-induced [Ca+2]i increase was isolated. These results demonstrate that high concentrations of Hcy can affect the mechanisms involved in [Ca2+]i regulation of HUVEC, and that alteration occurs specifically in the sustained phase, which has been directly associated with NO synthesis.