The aim of the report was to establish puncture techniques for the temporomandibular joint (TMJ) cavity in rats. The experimental sample comprised 30 male Sprague-Dawley rats. Under general anaesthesia the superior joint cavity of the rat was punctured either laterally (lateral puncture technique (LPT), n 5 11), anteriorly (anterosuperior puncture technique (ASPT), n 5 13) or anteroinferior puncture technique (AIPT), n 5 6) using a 27-gauge needle. After the tip of the needle was confirmed by micro-CT (R-mCT H , Rigaku, Tokyo, Japan) located on the mandibular fossa, 0.05 ml of contrast media was injected under micro-CT fluoroscopic guidance. After confirmation that the joint cavity was filled with contrast media, micro-CT imaging was carried out. The puncture for LPT was accurate in 5 of the 11 animals. The ASPT was accurate in all 13 animals. The AIPT punctured 3 of the 6 animals. Furthermore, the ASPT and AIPT demonstrated improved preservation of the needle; it was harder to detach the needle, which led to greater stability. These results suggest that ASPT assisted by R-mCT H is useful for basic research, including drug discovery and pathogenesis of TMJ diseases.