A genomic clone containing two linked human pregnancy-specific beta 1-glycoprotein (PS beta G) genes has been isolated and characterized. The two genes are arranged in the same 5'----3' orientation; the 3' region (including the A2 and B-C exons) of the upstream gene, PSGGA, is linked to the 5' region (including the 5'/L and L/N exons) of PSGGB, the downstream gene. Depending upon the domains compared, PSGGA and PSGGB share 92-98% nucleotide and 86-95% amino acid sequence identity with PSG93, the most abundant PS beta G transcript. The 3' exon (B-C) of PSGGA contains four alternative splice sites and three polyadenylylation sites, which account for the 3' heterogeneity previously reported in the PS beta G family. Each of the predicted PSGGA-encoded proteins would have a different carboxyl terminus. PSGGB corresponds to the previously identified cDNA PSG6, which encodes proteins containing a 34-amino acid leader peptide and a 108-amino acid N domain, which is one amino acid shorter than the majority of PS beta G N domains. Additionally, the PSGGB-encoded proteins contain the cell-surface recognition tripeptide Arg-Gly-Asp, shared by several previously reported PS beta Gs as deduced from cDNA sequences. Northern blot hybridization performed with a PSGGB-specific oligonucleotide probe to the N domain revealed that PSGGB or a PSGGB-like gene encodes a major 1.7-kilobase mRNA in hydatidiform mole tissues and a major 2.0-kilobase mRNA in term placenta tissues. Moreover, the PSGGB-specific probe hybridized most strongly with mRNA from molar trophoblastic tissue, suggesting that the PSGGB-like species may be the gene preferentially expressed in gestational trophoblastic disease. Additionally, the sequence of a 2315-base-pair PS beta G cDNA (PSG95) that contains an N-A1-A2-B2-C domain arrangement is reported. The coding region of PSG95 is identical to the previously reported cDNA clones PSG1d and FL-NCA, but PSG95 contains an additional 518 and 523 base pairs in the 3' end as compared with PSG1d and FL-NCA, respectively.