This study compared the threshold of pain sensitivity in the anterior mandibular mucobuccal fold with the posterior. This was followed by a comparison of the reduction of needle insertion pain in the anterior mucobuccal fold and the pterygo-temporal depression by either topical anesthesia or nitrous oxide inhalation. The pain threshold was determined by an analgometer, a pain-measuring device that depends on pressure readings; additionally, pain caused by a needle inserted by a normal technique was assessed using a visual analog scale (VAS). The threshold of pain was significantly lower in the incisor and canine regions than in the premolar and the molar regions (P < 0.001). Compared to a placebo, topical anesthesia significantly reduced the pain from needle insertion in the mucobuccal fold adjacent to the mandibular canine (P < 0.001), but did not significantly reduce pain in the pterygotemporal depression. The addition of 30% nitrous oxide did not significantly alter pain reduction compared to a control of 100% oxygen. These results suggest that topical anesthesia application may be effective in reducing the pain of needle insertion in the anterior mandibular mucobuccal fold, but may not be as effective for a standard inferior alveolar nerve block. The addition of 30% nitrous oxide did not lead to a significant improvement.