The objective of this study was to determine the effect of time on the clinical efficacy of topical anesthetic in reducing pain from needle insertion alone as well as injection of anesthetic. This was a randomized, double-blind, placebo-controlled, split-mouth, clinical trial which enrolled 90 subjects, equally divided into 3 groups based upon time (2, 5, or 10 minutes) of topical anesthetic (5% lidocaine) application. Each group was further subdivided into 2: needle insertion only in the palate or needle insertion with deposition of anesthetic (0.5 mL 3% mepivacaine plain). Each subject received drug on one side and placebo on the other. Subjects recorded pain on a 100-mm visual analog scale (VAS). The results showed that for needle insertion only, 5% lidocaine reduced pain as determined by a significant difference in mean VAS after 2 minutes (20.1 mm, P < .002), 5 minutes (15.7 mm, P < .022), and 10 minutes (13.7 mm, P < .04), as analyzed by paired t tests. For needle insertion plus injection of local anesthetic, a significant difference in mean VAS was noted only after 10 minutes (14.9 mm, P < .031), yet pain scores for both topical anesthetic and placebo were elevated at this time point resulting in no reduction in actual pain. Time of application did not result in a significant difference in effect for either needle insertion only or needle insertion plus injection of local anesthetic, as analyzed by 1-way analysis of variance (ANOVA). In conclusion, topical anesthetic reduces pain of needle insertion if left on palatal mucosa for 2, 5, or 10 minutes, but has no clinical pain relief for anesthetic injection.