Light transmission aggregometry (LTA) is used worldwide for the investigation of heritable platelet function disorders (PFD), but interpretation of results is complicated by the feedback effects of ADP and thromboxane A(2) and by the overlap with the response of healthy volunteers. Over 5 years, we have performed lumi-aggregometry to nine platelet agonists in 111 unrelated research participants with suspected PFDs and in 70 healthy volunteers. Abnormal LTA or ATP secretion test results were identified in 58% of participants. In 84% of these, the patterns of response were consistent with defects in Gi receptor signalling, the thromboxane pathway and dense granule secretion. Participants with defects in signalling to Gq-coupled receptor agonists and to collagen were also identified. Targeted genotyping identified three participants with function disrupting mutations in the P2Y(12) ADP and thromboxane receptors. Our study illustrates that detailed phenotypic analysis using LTA and ATP secretion is a powerful tool for diagnosis of PFDs and enables subdivision at the level of platelet signaling pathways and in some cases to individual receptors. We further demonstrate that most PFD can be reliably diagnosed using a streamlined panel of key platelet agonists and specified concentrations suitable for testing in most clinical diagnostic laboratories.