The influence of analgesic agents on neurons activated by stimulation of the temporomandibular joint (TMJ) region is not well defined. The spinomedullary junction [trigeminal subnucleus caudalis (Vc)/C(1-2)] is a major site of termination for TMJ sensory afferents. To determine whether estrogen status influences opioid-induced modulation of TMJ units, the classical opioid analgesic, morphine, was given to ovariectomized (OvX) rats and OvX rats treated for 2 days with low-dose (LE2) or high-dose (HE2) 17beta-estradiol-3-benzoate. Under thiopental anesthesia, TMJ units in superficial and deep laminae at the Vc/C(1-2) junction were activated by injection of ATP (1 mm) directly into the joint space. In superficial laminae, morphine inhibited evoked activity in units from OvX and LE2 rats in a dose-related and naloxone-reversible manner, whereas units from HE2 rats were not inhibited. By contrast, in deep laminae, morphine reduced TMJ-evoked unit activity similarly in all groups. Morphine reduced the background activity of units in superficial and deep laminae and resting arterial pressure similarly in all groups. Morphine applied to the dorsal surface of the Vc/C(1-2) junction inhibited all units independently of E2 treatment. Quantitative polymerase chain reaction and immunoblots revealed a similar level of expression for mu-opioid receptors at the Vc/C(1-2) junction in LE2 and HE2 rats. These results indicated that estrogen status differentially affected morphine modulation of TMJ unit activity in superficial, but not deep, laminae at the Vc/C(1-2) junction in female rats. The site(s) for estrogen influence on morphine-induced modulation of TMJ unit activity was probably outside the medullary dorsal horn.