Intracellular free calcium ([Ca<sup>2+</sup>]<sub>i</sub>) was measured in single cells of a confluent endothelial monolayer subjected to defined flow. Flow medium containing adenosine triphosphate (ATP) was used to study the influence of flow forces upon agonist-response coupling as mediated via the P2<sub>y</sub>-purinoceptor. [Ca<sup>2+</sup>]<sub>i</sub> responses were highly sensitive to the fluid motion at the cell surface; consecutive small increases of flow stimulated large [Ca<sup>2+</sup>]i transients with the levels returning to baseline at the new flow rate within 250 s. The characteristics of [Ca<sup>2+</sup>]<sub>i</sub> transients were also influenced by decreasing flow. Since potent ecto-nucleotidases at the endothelial cell surface rapidly degrade ATP, we postulated that a combination of flow and degradative enzymes regulates the mass transport of ATP in the boundary layer. The hypothesis predicts that step increases of flow exceed the capacity of the ectonucleotidases and allow ATP to reach the receptor. Experiments were conducted to compare ATP and ADP<sub>β</sub>S, a nonhydrolyzable ATP analog that resists degradation by surface ectonucleotidases, and calculations of ATP mass transport to the cell surface were compared to estimates of surface clearance rates. Calculations of mass transport coefficients for ATP in the boundary layer demonstrated that changes of flow which elicited a prominent [Ca<sup>2+</sup>]<sub>i</sub> response represented 26-73 % changes in the mass transport of ATP from the bulk fluid. When steady-state mass transport coefficients for ATP under various flow conditions were compared with the estimated rate constant for surface degradation of ATP, ratios close to unity were obtained. These results suggest that both boundary layer mass transport and ATP clearance rates can be rate-limiting for flow-mediated activation of the P<sub>2y</sub>-receptor. The experiments provide evidence for differential signal transduction responses in the endothelium driven by diffusion gradients (derived from both the blood and the vessel wall), which are likely to vary widely in the complex flow fields encountered in vivo.