Background: EMLA cream, a eutectic mixture of lidocaine and prilocaine, is a topical anesthetic, frequently used to avoid pain during venipuncture and superficial surgery. However, the depth of analgesia needs further exploration. Objective: Our purpose was to investigate the depth of cutaneous analgesia after application of EMLA cream. Methods: In a single-blind crossover study, EMLA cream was applied under occlusion to the left thighs of 16 subjects. Eleven had application times of 60 and 120 minutes. Five subjects had application times of 3 to 4 hours. Approximately 10 minutes after removal of the EMLA cream, a skin biopsy punch (diameter 4 mm) was inserted to a depth of 1 mm into the skin and the subject rated the pain intensity on a visual analogue scale. If there was no pain or if pain was assessed as acceptable, a new biopsy punch was inserted at a new site to the depth of 2 mm. In this manner, the insertion depth was gradually increased in steps of 1 mm down to a maximum of 6 mm. No skin specimens were removed. Results: After 60 and 120 minutes of EMLA application the mean insertion depths with acceptable pain were 2.9 and 4.5 mm (P < .01), respectively. After 3 to 4 hours of application, 6-mm deep insertions were made with acceptable pain in all 5 subjects. Conclusion: Skin biopsy punch insertions in steps of 1 mm appear adequate for assessing the depth of cutaneous analgesia. Biopsy punch insertions with acceptable pain can be made to depths of 1 to 2 mm after 60 minutes, to 2 to 3 mm after 120 minutes, and to 6 mm after 3 to 4 hours of EMLA cream application. (J Am Acad Dermatol 2000;42:584-8.)