The rat placental PRL family consists of molecules structurally similar to PRL and GH, and to date nine members have been identified. In the course of investigating late stage specific placental PRL family expression by differential display, we have isolated a complementary DNA encoding a new molecule that is highly homologous to PRL-like protein C (PLP-C) and PLP-D, and named this molecule PLP-H. The complementary DNA encoded a mature protein of 239 amino acids, including a 31-amino acid signal sequence. Sequence comparison between PLP-H and other members of the placental PRL family showed that PLP-H is highly homologous to PLP-C and PLP-D (78% and 67% homology at the amino acid level, respectively). Expression of PLP-H was similar to that of PLP-C and PLP-D; PLP-H messenger RNA (mRNA) first appeared on day 14 of pregnancy, and its expression increased until term. RT-PCR analysis showed that PLP-H as well as PLP-C and PLP-D are expressed in all rat strains examined, confirming that PLP diversity is not due to strain differences. In situ hybridization analysis indicated that PLP-H mRNA is specifically expressed in spongiotrophoblast cells and in trophoblast giant cells of the placental junctional zone. Differentiated Rcho-1 cells also expressed PLP-H mRNA, whereas undifferentiated Rcho-1 cells did not. PLP-H seems to exist as a secretory protein because its N-terminal sequence is identical to that of GH/PRL-like molecule secreted from placental explants. PLP-H contains two putative N-glycosylation sites and eight cysteine residues, of which six are highly conserved in the placental PRL family. We prepared a recombinant protein for PLP-H together with PLP-D using a COS7 transfection system. Purified PLP-H showed two bands with molecular masses of 27 and 29 kDa. Only the 27-kDa protein was detected after N-glycosidase treatment, indicating that PLP-H is a glycoprotein. PLP-H and PLP-D did not stimulate the proliferation of Nb2 lymphoma cells or the phosphorylation of Janus kinase-2 and signal transducer and activator of transcription-5. These data indicate that PLP-H and PLP-D are nonlactogenic hormones. Thus, we have cloned a new member of the PLP subfamily, PLP-H, which has features in common with PLP-C and PLP-D.