Our objectives were to determine lactational and reproductive outcomes in response to increased milking frequency (MF), injection of estradiol cypionate (ECP), and treatment with bovine somatotropin (bST). Lactating dairy cows (n = 144) were blocked by lactation number (1 vs. 2+) and assigned randomly to a 2 x 2 x 2 factorial experiment consisting of 8 treatment combinations: 1) MF consisting of 4x daily milking (4x) for the first 30 d in milk (DIM) vs. 2x daily milking (2x), with all cows milked 2x after 30 DIM; 2) 10 mg of ECP given postpartum at 8 +/- 3 DIM versus controls that received ECP diluent (oil); and 3) biweekly bovine somatotropin (bST), starting sometime after 60 DIM, versus no bST. Ovulation before the first artificial insemination was synchronized by using Heatsynch (GnRH injection 7 d before PGF2alpha followed in 24 h by ECP), and cows were artificially inseminated after detected estrus or at 48 h after ECP, whichever came first. Pregnancy was assessed by transrectal ultrasonography 28 to 30 d after artificial insemination. Daily yield and weekly components of milk were measured during the first 90 DIM. Intervals to first and second postpartum ovulation were unaffected by treatment, but cows were in estrus earlier after 2x (24 +/- 4 d) than 4x (41 +/- 4 d) daily MF, and sooner after ECP (25 +/- 3 d) than after oil (39 +/- 4 d) treatment. Pregnancy rates among 4x cows increased for ECP versus oil (52.8 vs. 27.8%) more than for cows with 2x MF treated with ECP versus oil (50.0 vs. 39.4%). Increased MF increased daily milk yields and energy-corrected milk yields during the first 30 DIM. Although milk yields were increased acutely by ECP during the 10 d after its injection, subsequent milk yields were decreased for ECP-treated cows previously milked 4x daily. Treatment with bST increased overall daily milk yields most in cows previously milked 2x daily and treated with oil and those milked 4x daily and treated with ECP. We concluded that early postpartum ECP injection increased pregnancy rates, but generally had detrimental effects on milk yields after 30 DIM for ECP-treated cows previously milked 4x daily, unless those cows also were treated with bST.