Objective: To describe the pain associated with diagnostic tympanocentesis and to gather preliminary data comparing the efficacy of 3 methods of pain reduction for tympanocentesis., Methods: In children 6 to 36 months of age undergoing tympanocentesis for acute otitis media, the authors measured pain and distress throughout all phases of the procedure and recovery using physiological (heart rate) and behavioral measures (cry duration, Global Mood Scale score, and pain visual analog scales). They compared--in a randomized controlled trial--3 pain reduction interventions: acetaminophen, acetaminophen plus codeine, and ibuprofen plus midazolam., Results: Heart rate increased throughout the procedure, peaking during needle aspiration. Children treated with acetaminophen alone had higher peak heart rates and Global Mood Scale scores during parts of the procedure., Conclusions: Acetaminophen alone may not be as effective in reducing pain-related physiological and behavioral changes as acetaminophen plus codeine or ibuprofen plus midazolam during diagnostic tympanocentesis.