We have isolated a cDNA clone for the 23-kDa mouse placental lactogen II (mPL-II) from a phage Agtll expression library containing cDNA synthesized from BALB/c placental RNA. Translation in vitro of placental mRNA selected by hybridization to the mPL-ll cDNA clones yields a 26-kDa polypeptide that is the size of the expected precursor protein and that is immunoprecipitated with anti-mPL-II antiserum. The mPL-ll cDNA clones hybridize to a 1.0-kilobase placentalspecific mRNA. This mRNA, found in the fetal portion of the placenta, appears as early as day 10 of gestation and increases to a maximal level by day 12. The mPL-ll cDNA nucleotide sequence has been determined. This sequence contains an open reading frame encoding a polypeptide of 222 amino acids with the amino-terminal 31 amino acids forming the signal sequence for secretion. The predicted secreted protein has 51% amino acid homology with mouse prolactin.