Electrical stimulation of the midbrain locomotor region (MLR) in 76 ovariectomized, urethan-anesthetized female rats elicited antidromic action potentials in 252 preoptic neurons. Thresholds and refractory periods for the activation ranged from 60 to 1550 microA and 1.3 to 5.0 ms, respectively. The probability distribution for the peak-to-peak amplitude (2-14 mV) or the overall duration (0.7-4.4 ms) was bell-shaped, whereas that for the latency (1.8-33.5 ms) was distinctively bimodal with a division at 12.0 ms. Two groups of preoptic neurons of a similar soma size therefore project to the MLR presumably via different routes. In 121 neurons with latencies =<12.0 ms, estrogen lowered the antidromic activation thresholds (nested analysis of variance, P<0.02), but 131 neurons with latencies > 12.0 ms had their thresholds increased (P<0.005) and refractory periods prolonged (P<0.02) by estrogen. Even though both overlapped in part, many potentials with the shorter latencies were recorded from the medial part of the lateral preoptic area (mLPO), lateral to the recording sites of the longer-latency potentials in the medial preoptic area (MPO). The observed antagonistic effects of estrogen on the two groups of preoptic neurons with axons to the MLR may contribute to increased locomotor activity in female rats in estrus.