The placenta secretes various factors in stage- and cell-specific manners. We have identified a cDNA encoding a novel protein with 124 amino acids, which was named spongiotrophoblast specific protein (SSP). SSP is highly homologous to mouse 4311, showing 81% and 59% similarity at the nucleotide and amino acid levels, respectively. Northern blot analysis showed that SSP mRNA was first detected on Day 14 of pregnancy, peaked on Day 16, and remained elevated until term. In situ hybridization analysis showed that SSP mRNA was specifically expressed in spongiotrophoblast cells of Day 20 placenta but not in Day 12 placenta. No expression was detected from the differentiated or undifferentiated rat choriocarcinoma Rcho-1 cell line. Native SSP was detected as a 19-kDa molecule by Western blotting in cell extracts prepared from the junctional zone. SSP was predicted to be a secretory protein, because 1) a hydropathy test revealed that SSP contained an N-terminal hydrophobic region and 2) native SSP was also detected in the cultured media of junctional zone explants. To further investigate a potential signal peptide of this protein, sets of recombinant SSP were generated using a COS7 transfection system. The N-terminal amino acid sequence of secreted recombinant SSP confirmed that the N-terminal 17 amino acids had been cleaved to produce a secretory protein. Thus, we have identified and cloned a novel secretory protein, SSP, which is specifically expressed by rat spongiotrophoblast cells during the latter half of pregnancy.