Parathyroid hormone (PTH)-mediated gene activation was assessed in the osteoblast-like rat cell line ROS17/2.8 with two PTH fragments harboring distinct activating domains: PTH-(1-34) and PTH-(28-48). The PTH response of genes expressed immediate early in the cell cycle or in the osteoblast developmental sequence was investigated. In addition, subtractive cloning was used to identify genes in ROS17/2.8 cells that are activated by the two PTH domains. PTH-(1-34) immediately increased the transcript levels of c-fos and c-jun at a considerably higher rate than PTH-(28-48). A significant immediate PTH effect on osteoblastic marker genes could not be detected, with the exception of elevated ornithine decarboxylase transcript levels. However, continuous application of PTH-(1-34) increased transcript levels of the osteoblast-specific osteocalcin gene and reduced those of other osteoblastic marker genes including alkaline phosphatase and the PTH/PTH-related peptide receptor. By subtractive cloning, nine cDNAs were isolated corresponding to mRNAs directly up-regulated by PTH-(1-34) or PTH-(28-48). Among these were a cyclic phosphodiesterase, a (cytosine 5)-methyltransferase, an 80-kDa protein kinase C substrate, junB, and a novel GC-binding protein. Three cDNAs are unknown at present. Interestingly, in all cases, the efficiency of gene activation by PTH-(28-48) was substantially lower in comparison with PTH-(1-34). PTH-mediated protein kinase C signaling in ROS17/2.8 cells may therefore constitute a minor pathway in comparison with the dominant cAMP/protein kinase A cascade.