Radioimmunoassays of estrogen and LH, and radioligand assays of progesterone were conducted on plasma samples collected serially and frequently during pregnant and nonpregnant cycles of Beagle bitches. Measurements of LH daily throughout proestrus and estrus in 20 cycles yielded mean (± SEM) basal levels of 1.4 ± 0.1 ng/ml, preovulatory elevations for 1-3 days, and peaks (7.5 ± 0.8 ng/ml) on the first day of estrus, or on the previous day. The day of the LH peak was designated as Day 0 for each cycle. Preovulatory increases in plasma progesterone could not be temporally dissociated from elevations in LH. Mean levels on Days -1, 0 and +1 were 0.8 ± 0.1, 1.6 ± 0.2 and 2.6 ± 0.2 ng/ml, both increases being significant (P<.001). Progesterone increased rapidly throughout estrus, reached 19.1 ± 2.5 ng/ml on Day 10 and a maximum of 22.9 ± 2.7 ng/ml on Day 25, and remained elevated until Day 30 at 19.9 ± 2.7 ng/ml. Mean plasma estrogen rose during proestrus; levels for Days 10, 3 and 1 before the LH peak were 26 ± 4, 43 ± 4, and 62 ± 4 pg/ml, respectively. Peak levels ranged from 46 to 113 ng/ml and occurred 0, 1, or 2 days prior to the LH peak. Estrogen levels fell rapidly coincident with or immediately following the LH peak.