The calcium and phospholipid-dependent protein kinase C (PKC) system appears to play an important role in mediating hormonal effects in various tissues including bone. Accordingly, we characterized PKC activity in the UMR-106-01 rat osteosarcoma osteoblastlike cell line and examined its hormonal regulation. UMR-106-01 cells were found to possess a classic, phorbol ester-activated PKC system, which was highly calcium and phospholipid dependent. A 30 s exposure to 10 nM bovine parathyroid hormone (PTH) (1-34) increased cytosolic and membrane-bound PKC activity by 12 and 157%, respectively, resulting in a 2.2-fold increase in the membrane-bound to cytosolic (MB/C) activity ratio (all p less than 0.01). The MB/C activity ratio was highest at 20 min, exhibiting a 2.8-fold increase over the control values (p less than 0.01). In contrast, 10 nM insulin increased cytosolic PKC activity but decreased membrane-bound activity, resulting in a 61% decrease in the MB/C activity ratio at 20 min (p less than 0.02). Moreover, insulin reduced PTH stimulation of the PKC activity ratio by 42 and 62% at 30 s and 20 min, respectively (p less than 0.02). Thus, PTH and insulin have opposing effects on the PKC activity ratio in UMR-106-01 cells.