To further understand the mechanism of PTH effects on bone and bone cells, we have analyzed the effect of PTH on specific protein phosphorylation in cells isolated from neonatal mouse calvaria. Four populations of cells (I-IV), isolated by sequential digestion with chromatographically purified bacterial collagenase isozymes and neutral proteinase, were cultured overnight. Alkaline phosphatase activity was greater than acid phosphatase activity in all four populations. PTH stimulated cyclic AMP production in all four populations, although the effect was greatest in populations II and III. Cultured cells were treated with PTH for up to 15 minutes. Cytosolic and membrane fractions were obtained and assayed for in vitro protein phosphorylation. No hormonal effects were found in membrane fractions. In cytosol fractions, treatment of the population II cells for 10-15 minutes with 0.1 microM PTH decreased the subsequent protein phosphorylation of an 85,000 Mr protein. In contrast, PTH treatment increased in vitro phosphorylation of both the 85,000 and 35,000 Mr proteins in population III cells. Phosphorylation of the 35,000 Mr protein was cyclic AMP-dependent. All of the phosphoproteins appeared to be phosphorylated solely on serine or threonine residues except the 85,000 Mr protein which may also contain significant amounts of phosphotyrosine. Therefore, some of the effects of PTH are cyclic AMP-mediated and other effects may be mediated through tyrosine phosphorylation. These data indicate that PTH has differential effects on in vitro protein phosphorylation in two separable populations of isolated neonatal mouse calvarial cells and support a hypothesis that multiple osteoblastlike cells exist in vivo.