The endothelium is the innermost layer of all blood vessels and changes in the behaviour of this single layer of cells underlie almost all cardiovascular disease. To regulate cardiovascular function, the endothelium must integrate hundreds of signals. These signals can arrive from substances circulating as well as neighbouring cells. However, how the endothelium detects and relays the information stored in each of these signals to initiate a physiological output is not fully understood. The problem in integrating signals is made even more complex as the central relay system within an endothelial cell is a single ion, Ca2+. Here, we show the endothelium manages a multitude of extracellular signals by utilising spatially-distinct endothelial cells. Furthermore, the Ca2+ signal generate by each activator has unique distinguishing characteristics. To determine how multiple extracellular signals are detected in the endothelium we analysed the Ca2+ activity from ~1000 endothelial cells from intact arteries. Male-Sprague-Dawley (150-250g) rats were euthanized by CO2 overdose (Schedule 1; Animals (Scientific Procedures) Act 1986) and second order mesenteric arteries were extracted, cut open and pinned flat. The endothelium was loaded with a Ca2+ indicator Cal-520/AM and imaged using fluorescence microscopy. Ca2+ activity was separately analysed in each of the 1000 individual endothelial cells using a custom built software package written in Python. For each activator (ACh, ADP, ATP & Histamine) full concentration response curves were carried out to determine the EC25 concentration (the concentration at which 25% of cells responded). There was variation in the EC25 for the four agonists (ACh = 15.6 nM; ADP = 119.8 nM; ATP = 1.2 mM; Histamine = 6.3 mM). The addition of the four activators (applied separately) at their respective EC25 concentration, to the same field of endothelium revealed the endothelium utilises sub-populations of endothelial cells to detect each activator. Endothelial cells were organised in clusters and Ca2+ signals propagated between cells as Ca2+ waves. The cells responsible for the detection of a specific activator were spatially unique and there was no significant overlap with cells that were primed to detect another agonist. For example, of the 25% of cells that responded to ACh only 21.5% responded to ADP, 19.6% responded to ATP and 18.5% responded to Histamine (p<0.05, n=5). There was no significant overlap in the sensitivity of the endothelium to the different agonists for all the various permutations (ADP vs ACh, ATP & Histamine; ATP vs ACh, ADP & Histamine; Histamine vs ACh, ADP, ATP; p<0.05, n=5). Each activator generated a unique Ca2+ signal that was specific to the agonist. By using spatially-distinct cells, unique signal characteristics and communication, the endothelium is able to detect and relay the information stored in the multitude of extracellular activators to initiate physiological outputs.