Members of the large rat prolactin gene family located on chromosome 17, are expressed in one or more placental trophoblast cell types and in maternal decidua, at specific times during pregnancy. Studies to identify the factors involved in these highly specific developmental expression patterns, using limited amounts of 5' flanking DNA, have met with only partial success. Here we report the isolation and characterization of an 80 Kb rat genomic clone, P1 12830, containing linked rPLII, rPLP-I, and rPLP-B genes with substantial amounts of 5' and 3' flanking DNA, as well as a rPLII-related pseudogene, the first to be described in this gene family. This clone was used to create F0 transgenic mice and the levels of expression of the three rat genes were compared with those of the endogenous mouse genes, using RT-PCR. Each rat gene was expressed differently in the same placenta, confirming the importance of sufficient flanking sequences in the expression of the individual genes. These studies emphasize the need for large genomic clones in defining the complete complement of factors that regulate the developmental expression of the rat PRL gene locus.