Abstract Immunoreactivity for α4, β1 and δ GABA A receptor subunits on neurones in the periaqueductal gray matter was investigated at different stages of the estrous cycle in Wistar rats. Immunostaining for α4, β1 and δ GABA A receptor subunits was present on neurones throughout the periaqueductal gray matter. The numbers of subunit-immunoreactive neurones remained constant during the early phases of the estrous cycle (proestrus to early diestrus) but increased significantly in late diestrus. Dual immunolabeling for the GABA synthesizing enzyme glutamic acid decarboxylase revealed that almost 90% of the subunit-positive cells contained immunoreactivity for glutamic acid decarboxylase. During the early phases of the estrous cycle (proestrus to early diestrus), approximately one third of the glutamic acid decarboxylase-positive population co-localized α4, β1 and δ GABA A receptor subunits. When the number of subunit positive cells increased during late diestrus, the proportion of the glutamic acid decarboxylase-containing population that expressed α4, β1 and δ GABA A receptor subunits almost doubled. We propose that GABA A receptors with the α4β1δ configuration are expressed by GABAergic neurones in the periaqueductal gray matter and that the numbers of cells expressing these subunits are increased in late diestrus in line with falling plasma progesterone levels. Changes in GABA A receptor expression may lead to changes in the excitability of the neural circuitry in the periaqueductal gray matter.