Intermittent PTH therapy is currently the only anabolic therapy for osteoporosis. As the mineralisation of the extracellular matrix of bone is essential for normal function it is vital that the effects of PTH on key regulators of mineralisation are uncovered. Ablation of Alpl, Phospho1 or Smpd3 results in skeletal hypomineralisation and as such this study examined the effects of bovine (b)PTH 134 on their expression. MC3T3 (clone-14) osteoblast-like cells display temporal increases in Phospho1, Alpl and Smpd3 expression (150, 60 and 60-fold respectively by day 10; P<0.001). At day 10, Phospho1 mRNA was significantly reduced after a 15 min exposure to bPTH (50 nM; 80% decrease; P<0.001) Further reductions were evident after 1 and 6 h bPTH exposures (96 and 93% decrease respectively; P<0.001) and persisted to 24 h (48% decrease; P<0.05). Smpd3 expression was similarly reduced after 6 and 24 h exposures (97 and 91% respectively; P<0.001). PHOSPHO1 and SMPD3 protein was markedly reduced after 24 and 48 h bPTH treatment. In contrast, Alpl mRNA levels increased after 1 (2.8-fold; P<0.05) and 6 h (3.6-fold; P<0.001) bPTH exposure which was consistent with increased TNAP protein after 24 and 48 h bPTH treatment. Phospho1, Smpd3 and Alpl showed dose dependent (0.0550 nM) responses to 24 h bPTH treatment. Indeed, a 50% reduction (P<0.001) in Phospho1 and Smpd3 expression was achieved with 0.5 nM bPTH with comparable changes at the protein level. Induction of Alpl mRNA and protein was achieved with 5 nM bPTH. The cAMP activator forskolin induced a suppression of Phospho1 comparable to the effects of bPTH (93 and 96% respectively). Forskolin stimulated Alpl expression (2.4-fold; P<0.05). The suppression of Phospho1 by bPTH was partially obstructed by the PKA-inhibitor, PKI 524 (45% reduction compared to 75% observed in bPTH only cultures). In summary, bPTH shows potent effects on PHOSPHO1, SMPD3 and TNAP during osteoblast-mineralisation with initial studies implicating the cAMP/PKA signalling pathway.