Abstract: Lidocaine is an amide type local anaesthetic and diphenhydramine is a first-generation antihistamine drug. The aim of the present study was to investigate the influence of diphenhydramine co-administration on the extent of lidocaine binding to rat serum and maxillofacial tissue proteins in order to determine a possible synergistic action that could enhance lidocaine’s anaesthetic/analgesic activity. Twenty-eight Wistar rats divided in 4 groups (I, II, III, IV) received lidocaine in the masseter muscle. Groups II and IV received diphenhydramine per os 2 h before lidocaine administration. Groups I and II were sacrificed after 15 min and groups III and IV 30 min after lidocaine injection. Masseter and mandible samples were isolated and incubated in NaCl 0.9% solution while serum was obtained through blood centrifugation. Free lidocaine fraction in the tissues’ incubation medium and the serum was obtained through ultrafiltration and determined by radioscopic method in a β-counter Lidocaine’s free fraction levels (μg g-1) were enhanced after 15 min under diphenhydramine co-administration in all samples from (7.1319±1.4066)x10-4 to (12.1097±3.7528)x10-4 in serum, from 0.9339±0.3077 to 2.6791±1.1648 in masseter (p<0.01) and from 0.3898±0.0879 to 0.6918±0.2743 in mandible (p<0.05). A statistically significant increase in free anaesthetic levels was also noticed after 30 min in serum from (8.6227±0.6902)x10-4 to (13.9518±4.9849)x10-4 (p<0.05). Lidocaine’s increase could probably be attributed to mechanisms influencing its protein binding properties. Consequently, a possible synergistic action of the two drugs’ combination is demonstrated which could enhance lidocaine’s anaesthetic action, affecting depth and duration of anaesthesia.