Cardiac transplant recipients often anticipate and suffer varying degrees of discomfort during surveillance endomyocardial biopsy (EMBx). We performed a randomized, blinded, placebo-controlled trial to determine whether topical anesthetic was associated with reduced pain and to identify factors associated with increased pain perception during EMBx. In 225 EMBxs, use of the eutectic mixture of lidocaine and prilocaine (EMLA) decreased pain score (-7.3 compared with placebo; p = 0.04); the other significant predictors of increased pain scores were time to achieve access (+2.3 per minute; p = 0.001) and female gender (+12.7 compared with males; p = 0.003). Topical anesthetic cream is associated with decreased pain during EMBx, even after adjusting for other predictors of pain, including female gender and longer time to achieve access. A better understanding of the factors affecting pain during EMBx could improve the comfort level of this procedure.