Our objectives were to determine pregnancy rates after altering times of the second GnRH injection, insemination, or both in a combined Presynch + Ovsynch protocol, to accommodate once-daily lockup of dairy cows. Lactating dairy cows (n = 665) from 2 dairy herds in northeastern Kansas were studied. Cows ranged from 24 to 44 d in milk (DIM) at the start of the Pre-synch protocol, which consisted of 2 injections of PGF(2alpha) 14 d apart, with the second injection given 12 d before initiating the Ovsynch protocol. Cows were blocked by lactation number and assigned randomly to 3 treatments consisting of variations of the Ovsynch protocol. Cows in 2 treatments received injections of GnRH 7 d before and 48 h (G48) after the PGF(2alpha) injection. Timed AI (TAI) was conducted at the time of the second GnRH injection (G48 + TAI48) or 24 h later (G48 + TAI72). Cows in the third treatment received the injections of GnRH 7 d before and at 72 h after PGF(2alpha) and were inseminated at the time of the second GnRH injection (G72 + TAI72). Pregnancy was diagnosed weekly by palpation per rectum of uterine contents on d 40 or 41 after TAI. Pregnancy rates differed between herds, but they were consistently greater for G72 + TAI72 than for G48 + TAI48 and G72 + TAI72. Subsequent calving rates were consistent with differences in initial TAI pregnancy rates. Pregnancy loss was least for cows on the G72 + TAI72 treatment. Body condition scores (BCS) ranged from 1.0 to 4.0 when assessed on Monday of the breeding week. An interaction of BCS and herd was detected in which cows in herd 1 having poorer BCS (<2.25) had greater pregnancy rates than cows of greater BCS (>/=2.25), whereas the reverse was true in herd 2 in which overall pregnancy rates were greater. We concluded that inseminating at 48 or 72 h after PGF(2alpha), when GnRH was administered at 48 h after PGF(2alpha), produced fewer pregnancies than inseminating and injecting GnRH at 72 h after PGF(2alpha) for cows whose estrous cycles were synchronized before initiating this variant of the Ovsynch protocol.