This study characterized the peripheral plasma bovine pregnancy-associated glycoprotein (bPAG) profile throughout gestation and examined the effect of stage of gestation and fetal number on this profile in Holstein cows after non-surgical embryo transfer. Cows (n = 12) were divided into three groups: group 1 = normal singleton pregnancies (n = 5); group 2 = normal twin pregnancies (n = 5); group 3 = abnormal twin pregnancies (n = 2). Blood was collected about every third day from day 0 (defined as the first day of standing estrus), then daily for the last 10 days of gestation, and sampling was stopped one day postpartum. The time-related changes in plasma bPAG concentrations were significantly (P < 0.01) affected by the stage of gestation and fetal number (P < 0.01), except during the last 10 days of gestation. In both normal pregnancy groups, bPAG concentration increased rapidly during the first trimester (0.5 +/- 0.1 to 14.6 +/- 1.7 ng/ml and 1.0 +/- 0.6 to 21.8 +/- 4.8 ng/ml, in singleton and twin-bearing groups respectively), then progressively between days 160 and 20 prepartum (31.6 +/- 6.2 to 114.3 +/- 31.3 ng/ml and 41.6 +/- 7.4 to 155.8 +/- 36.6 ng/ml in singleton and twin-bearing cows respectively). The mean concentration between days 20 and 10 prepartum approximately tripled (P < 0.001) in both these groups of cows (114.3 +/- 31.1 to 493.0 +/- 75.3 ng/ml and 155.8 +/- 36.6 to 409.3 +/- 114.7 ng/ml in singleton and twin-bearing cows respectively), but between days 10 prepartum and parturition the values increased about threefold (P < 0.01) in the singleton group (493.0 +/- 75.3 to 1352.8 +/- 286.5 ng/ml) and fivefold (P < 0.001) in the twin-bearing group (409.3 +/- 114.7 to 2154.0 +/- 505.7 ng/ml). The two cows in group 3 that gave birth prematurely to a stillborn calf or to a schistosomus reflexus calf exhibited an aberrant bPAG profile. Our results indicate that peripheral bPAG concentrations are correlated to the stage of gestation and fetal number, and that the profile of the peripheral plasma concentrations provides a useful indication of the feto-placental status.