New bone formation is associated with an increase in blood flow by the invasion of capillaries. Endothelial cells that line the capillaries can produce paracrine factors that affect bone growth and development, and in turn, could be affected by products produced by bone cells, in particular the osteoblasts. Since osteoblasts produce prostaglandins E, and F , , (PGE,, PGF, , ) , it was investigated if these PGs were agonists to bone-derived endothelial cells (BEE) by assessing changes in cAMP and free cytosolic calcium concentration ([Ca2'li) second messenger generation. We found that confluent cultures of BEE cells, a clonal endothelial cell line derived from bovine sternal bone, responded to 1 KM PGE, by an increase in CAMP. PGF, , at the same concentration was less potent in stimulating an increase in cAMP production in confluent BEE cells. Subconfluent cells with a morphology similar to that of fibroblastic cells were not as sensitive to PGE,-stimulated cAMP generation. PGF, , failed to elicit any cAMP production in subconfluent cultures. PGE, and PGF, , both stimulated an increase in [Ca2']i concentration in a dosedependent manner. The potency of PGE, was similar to that of PGF, , in stimulating an increase in [Ca"]i. The Ca2+ response was mostly independent of extracellular Ca+, was unchanged even with prior indomethacin treatment, was unaffected by caffeine pretreatment, but was abolished subsequent to thapsigargin pretreatment. The PC-induced increase in [Ca'+]i was also dependent on the confluency of the cells. In a subconfluent state, the responses to PGE, or PGF, , were either negligible, or only small increases in [Ca2+]i were noted with high concentrations of these two PGs. Consistent, dose-dependent increases in [Ca2+] i were stimulated by these PGs only when the cells were confluent and had a cobblestoned appearance. Since it was previously demonstrated that BEE cells respond to parathyroid hormone (PTH) by the production of CAMP, we tested if bovine PTH(1-34) amide [bPTH(1-34) also increased [Ca2+]i in these cells. No change in [Ca2']i was found in response to bPTH (1-34), although bPTH (1-34) stimulated a nine to tenfold increase in CAMP. We conclude that BEE cells respond to PGE, and PGF, , but not to bPTH(1-34) by an increase in [Ca2'li probably secondary to stimulation of phospholipase C and that the cAMP and [Ca2+]i second messenger responses in BEE cells are dependent on the state of confluency of the cells.