Prostatic cancer is frequently associated with new bone formation although the tumor-derived factors responsible for changes in bone cell function have not been identified. We have examined the synthesis of osteoblast-stimulating factors in a cultured human prostatic cancer cell line (PC-3) and show that conditioned medium from PC-3 cells stimulate mitogenesis and alkaline phosphatase in cells with the osteoblast phenotype (cultured rat osteosarcoma cells) and collagen synthesis in fetal rat calvaria. In order to characterize tumor-derived gene products which stimulate cells of the osteoblast phenotype messenger RNA (mRNA) was isolated from PC-3 cells and microinjected into Xenopus laevis oocytes. mRNA-directed translation products which were secreted into the oocyte medium were collected and assayed for a number of osteoblast stimulating properties. Translation products from PC-3 mRNA-injected oocytes stimulated division of cultured osteosarcoma cells by 8-fold and increased DNA synthesis as measured by incorporation of [3H]thymidine into these cells. In addition, tumor-derived translation products stimulated the production of alkaline phosphatase activity, a marker enzyme for bone formation, in cultured osteosarcoma cells. Oocytes injected either with water or with mRNA from a tumor not associated with bone formation were devoid of these activities. Total mRNA from the human prostatic cancer cells was then denatured and fractionated by size by agarose gel electrophoresis. When individual fractions of mRNA were eluted from the gel, translated in Xenopus oocytes, and the secreted translation products were tested for alkaline phosphatase-stimulating activity on osteoblast-like cells, the majority of the activity could be recovered in a mRNA fraction which was approximately 1800 bases in length. These results indicate that the PC-3 prostatic cancer cell line synthesizes a mRNA of approximately 1800 bases which codes for a heretofore unrecognized osteoblast-stimulating factor.