The pregnancy-associated glycoproteins (PAG) constitute a large family of recently duplicated genes. They show structural resemblance to pepsin and related aspartic proteinases. A total of 21 bovine (bo) PAG and 9 ovine (ov) PAG cDNA have been identified. Phylogenetic analysis indicated that the PAG are divided into two main groupings that accurately reflect their tissue expression, as determined by in situ hybridization. In the first pattern, represented by ovPAG-2 and boPAG-2, -8, -10, and -11 (where the numbering is arbitrary and reflects order of discovery within species), expression occurred throughout the outer epithelial layer of the placenta (trophectoderm). The second pattern was predominant localization to binucleate cells. Ribonuclease protection assays, which allow discrimination between closely related transcripts, have shown that the expression of PAG varies in a temporal manner over pregnancy. Of those bovine PAG expressed predominantly in binucleate cells, boPAG-1, -6, and -7 are expressed weakly, if at all, by Day 25 placenta, but are present at the middle and end of pregnancy. Others, such as boPAG-4, -5, and -9, are expressed at Day 25 and at earlier stages. Although not among the earliest PAG produced by the trophoblast, boPAG-1 has been used for pregnancy diagnosis, particularly in dairy cows, where there is a major need for a sensitive method capable of detecting pregnancy within 1 mo of conception. It seems likely that some of the newly discovered PAG will be better candidates than PAG-1 for pregnancy diagnosis.