1. Using synthetic proteinase-activated receptor-2 (PAR(2))-activating peptides (PAR(2)APs) corresponding to the tethered ligand domain of the extracellular N-terminus of PAR(2) to mimic the actions of activating proteinases and using primary cultures of calvarial osteoblasts derived from both wild-type (WT) and PAR(2)-null (KO) mice, we investigated the potential role of PAR(2) in regulating osteoblast function. 2. Primary calvarial osteoblasts from WT and KO mice were evaluated for their growth kinetics and mineralization in the absence of PAR(2) agonists and for their responses in a variety of functional assays to the PAR(2)APs Ser-Leu-Ile-Gly-Arg-Leu-amide (SLIGRL-NH(2)) and 2-furoyl-Leu-Ile-Gly-Arg-Leu-Orn-amide (2-fLIGRLO-NH(2)), as well as to trypsin. 3. In contrast with WT cells, PAR(2)-KO osteoblasts did not exhibit increased collagen Type I mRNA expression in response to SLIGRL-NH(2). When grown in serum-containing medium, KO cells increased in number more rapidly than WT cells, an effect that could be attributed to decreased apoptosis rather than increased proliferation. Surprisingly, in both WT and KO osteoblasts, the two PAR(2)APs induced mobilization of intracellular calcium stores. Similarly, the PAR(2)APs inhibited serum deprivation-induced apoptosis and parathyroid hormone-, 1,25-dihydroxyvitamin D(3)- or interleukin-11-induced mineralization in WT and KO cells. 4. We conclude that PAR(2) plays a role in osteoblast survival and collagen Type I mRNA induction and that osteoblasts can respond to the PAR(2)APs via both PAR(2)-dependent and -independent mechanisms.