We have cloned and characterized the gene and complementary DNA for a new kringle-containing protein. Although the function of this protein is not known, it has been called hepatocyte growth factor-like protein because it shares the same structural domains as hepatocyte growth factor, with four kringle structures followed by a region homologous to serine proteases. To determine if hepatocyte growth factor-like protein is synthesized by the same cells as hepatocyte growth factor, we surveyed adult mouse and developing mouse embryo tissues by in situ hybridization analysis using radiolabeled RNA for hepatocyte growth factor-like protein. Results of in situ hybridization analysis of adult mouse tissues show that among all surveyed tissues specific signal was restricted to the liver. Higher magnification of liver sections shows that hepatocytes were the only cell type expressing messenger RNA for hepatocyte growth factor-like protein, contrary to the reported presence of hepatocyte growth factor messenger RNA in fat-storing cells. A similar liver-restricted and hepatocyte-specific pattern of messenger RNA expression was observed in the developing mouse embryo at 14 days of gestation. All other developing tissues that were analyzed, as well as liver hematopoietic cells, did not express messenger RNA for hepatocyte growth factor-like protein at levels detectable by this technique. We conclude that although structurally similar to hepatocyte growth factor, hepatocyte growth factor-like protein is a novel protein synthesized primarily in hepatocytes.