Introduction: ATP plays an important role in the signalling mechanisms that regulate vascular tone and blood flow. We have previously shown both in vivo and in vitro (1,2) that increases in temperature during exercise or with passive heating, a three-fold increase in the release of ATP is stimulated from erythrocytes that was closely associated with the increases in muscle temperature. This source of ATP could also be derived from the endothelial cells lining the blood vessel walls. We therefore investigated the effect of both heating and cooling on cultivated human umbilical vein endothelial cells (HUVECs). Methods: HUVECs were exposed to a range of temperatures, the highest of which is known to increase erythrocyte ATP release at 39°C and compared this to 37°C (control – normal cell culture conditions) and 23°C (cool – room temperature). Confluent cultures grown on six-well plates were washed and incubated in Hanks balanced salt solution with 25 mmol l-1 HEPES, pH 7.35 containing 100 µmol l-1 ARL67156 an ecto-ATPase inhibitor. After an equilibration period, cells were incubated for 30 min in water baths set at 39°C and 37°C and room temperature (23°C). ATP release from HUVECs was measured in the supernatant at regular intervals for 30 min using a luciferin-luciferase assay at the end of which both cell numbers and protein content were determined. All data are mean ± SEM from three primary HUVEC cultures from different individuals sampled in duplicate and were compared using RM-ANOVA. Results: Initial ATP in the media was 49 ± 8 nmol l-1, after 30 min exposure of cells to 39°C or 23°C compared to control (37°C), ATP was 20 ± 5 nmol l-1 with heating, remained unchanged at 23°C (46 ± 14 nmol l-1), while in control conditions was 29 ± 9 nmol l-1 (all non-significant). To account for any detrimental effects on cell count or protein content caused by the changes in temperature, initial cell density was 11 ± 1 x 104 cells ml-1 while after 30 min there were 7 ± 1, 9 ± 2 and 9 ± 1 x 104 cells ml-1, whilst ATP normalised to total protein was 0.8 ± 0.1, 1.1 ± 0.3 and 0.6 ± 0.2 nmol mg-1 protein at 39°C, 23°C compared to 37°C respectively (all non-significant). Conclusion: Contrary to the profound effects of changes in temperature on ATP release from human erythrocytes (1), the current study suggests that human endothelial cells do not release ATP through a mechanism that is sensitive to physiologically hot and cool temperatures.