BACKGROUND: Adenosine-5’-triphosphate (ATP) is a neurotransmitterand inflammatory cytokine implicated in the pathophysiology of lowerurinary tract disease. ATP additionally reflects microbial biomassthus has potential as a surrogate marker of urinary tract infection(UTI). The optimum clinical sampling method for ATP urinalysis hasnot been established. We tested the potential of urinary ATP in theassessment of lower urinary tract symptoms, infection and inflammation,and validated sampling methods for clinical practice. METHODS: Aprospective, blinded, cross-sectional observational study of adultpatients presenting with lower urinary tract symptoms (LUTS) andasymptomatic controls, was conducted between October 2009 and October2012. Urinary ATP was assayed by a luciferin-luciferase method, pyuriacounted by microscopy of fresh unspun urine and symptoms assessedusing validated questionnaires. The sample collection, storage andprocessing methods were also validated. RESULTS: 75 controls and340 patients with LUTS were grouped as without pyuria (n = 100),pyuria 1-9 wbc $μ l(-1)$ (n = 120) and pyuria $≥$10 wbc $μl(-1)$ (n = 120). Urinary ATP was higher in association with femalegender, voiding symptoms, pyuria greater than 10 wbc μl(-1) and negativeMSU culture. ROC curve analysis showed no evidence of diagnostictest potential. The urinary ATP signal decayed with storage at 23°Cbut was prevented by immediate freezing at ≤ -20°C, without boricacid preservative and without the need to centrifuge urine priorto freezing. CONCLUSIONS: Urinary ATP may have a role as a researchtool but is unconvincing as a surrogate, clinical diagnostic marker.