We have shown recently that the medial preoptic area (MPO) robustly innervates discrete columns along the rostrocaudal axis of the midbrain periaqueductal gray (PAG). However, the location of PAG neurons responsive to MPO activation is not known. Anterograde tract tracing was used in combination with Fos immunohistochemistry to characterize the MPO -+ PAG pathway anatomically and ,functionally within the same animal. Focal electrical or chemical stimulation of MPO in anesthetized rats induced extensive Fos expression within the PAG compared with sham controls. Fos-positive neurons were organized as 2-3 longitudinal columns. The organization and location of these columns overlapped remarkably well with the distribution of fibers and terminals in PAG labeled by Phaseolus vulgaris leucoagglutinin (PHA-L) injected into the same MPO stimulation site. This indicates that MPO inputs may terminate on the soma or proximal dendrites of neurons exhibiting elevated Fos. A second series of experiments investigated whether MPO stimulation excited PAG neurons with descending projections to the medulla. Retrograde labeling of PAG neurons projecting to the medial and lateral regions of the rostroventral medulla (RVM) was combined with MPO-induced Fos expression. The results showed that a substantial population (3743%)of Fos-positive PAG neurons projected to the ventral medulla. This indicates that MPO stimulation engages PAG-medullary output neurons. Taken together, these results suggest that the MPO + PAG + RVM projection constitutes a functional pathway. This circuit may coordinately regulate neuroendocrine, motor, and autonomic adjustments necessary for the elaboration of sexual behaviors.Key words: reproduction; antinociception; cardiovascular regulation; brainstem; sexual behavior; immunohistochemistryThe medial preoptic area (h/[PO) is a sexually dimorphic structure Simerly et al., 1984;Bloch and Gorski, 1988) that plays a pivotal role in sexual behavior and neuroendocrine function (Lisk, 1966;Powers and Valenstein, 1972;Pfaff and Sakuma, 1979;Hansen et al., 1982;Arendash and Gorski, 1983;Kalra and Kalra, 1983;Docke et al., 1984;Sachs and Meisel, 1988;Simerly et al., 1990;DonCarlos et al., 1991;Takeo et al., 1993;Hoshina et al., 1994). We recently reported that MPO robustly innervates the midbrain periaqueductal gray (PAG) (Rizvi et al., 1992) and terminates in discrete, longitudinally organized columns running through the rostrocaudal axis of PAG.PAG plays a key role in antinociception (Reynolds, 1969;Liebeskind et al., 1973;Oliveras et al., 1974;Behbehani and Fields, 1979; Lovick, 3985;Morgan and Liebeskind, 1987;Reichling et al., 1988;Lovick, 1990), cardiovascular control (Lovick, 1985;Carrive et al., 1987;Carrive et al., 1988;Carrive et al., 1989a;Lovick, 1990;Verberne and Guyenet, 1992;Murphy et al., 1994a;Murphy et al., 1995), and many of the same neuroendocrine and reproductive functions as MPO (Pfaff and SchwartzGiblin, 1988;Ogawa et al., 1991;Shipley et al., 1995). PAG neurons involved in these functions also are ...