The central site of action and the cellular mechanism by which progesterone stimulates respiration were studied in ovariectomized cats that were anesthetized, paralyzed, and ventilated and in which respiratory sensory feedback mechanisms were either eliminated or controlled. Phrenic nerve activity served as an index of central respiratory output. Progesterone did not stimulate respiration in ovariectomized cats not pretreated with estrogen. In contrast, repeated doses of progesterone (0.1-1.0 microgram/kg, iv, cumulative) caused a sustained (greater than 45 min) dose-dependent facilitation of phrenic nerve activity in animals primed 3 days before study with 17 beta-estradiol (20 micrograms/kg, sc). Estrogen exposure is, therefore, a prerequisite for the respiratory response to progesterone in ovariectomized cats. This estrogen-dependent respiratory response to progesterone was attenuated in animals pretreated with either the estrogen receptor antagonist CI628 or the progesterone receptor antagonist RU486, indicating that the respiratory response is mediated by both estrogen and progesterone receptors. Inhibitors of protein (anisomycin) and RNA (actinomycin-D) synthesis caused a diminution of the respiratory response to progesterone, implicating a requirement for gene expression in the response. Midcollicular decerebration (which removed the diencephalon) attenuated, whereas decortication (which spared the diencephalon) did not affect the respiratory response to progesterone. Thus, the diencephalon appears to be a critical neuroanatomical substrate for the response. These results indicate that the respiratory response to progesterone is mediated, at a hypothalamic site, via a genomic mechanism with characteristics consistent with the prototypic mechanism for progesterone actions.