1. The aim of the study was to characterize the effects of hypoxia on agonist-stimulated phospholipase D (PLD) and phospholipase C activity of sheep pulmonary artery cultured smooth muscle cells. 2. Endothelin-1 (ET-1), 5-hydroxytryptamine (5-HT) and the protein kinase C (PKC) activator tetradecanoylphorbol acetate (TPA), stimulated a time-and concentration-dependent increase in [3H]-phosphatidylbutanol accumulation. This was abolished by pretreatment of the cells with the PKC inhibitor, Ro-318220, suggesting that agonist-stimulated phospholipase D activity is dependent upon the activation of PKC. 3. Hypoxia (PO2 20 mmHg for 30 min) stimulated basal [3H]-phosphatidylbutanol accumulation by approximately 2 fold and this activity was abolished by preincubation of the cells with 10 microM Ro-318220. 4. In cells preincubated in low O2 containing medium for 30 min, the subsequent agonist-stimulated accumulation of [3H]-phosphatidylbutanol was reduced. However, the decrease in stimulation was greater for ET-1 and 5-HT than for TPA. 5. ET-1 and TPA stimulated a time-dependent increase in protein kinase C-mediated psuedosubstrate phosphorylation. Following preincubation for 30 min in low O2 containing media, basal pseudosubstrate phosphorylation increased whilst the fold stimulation by TPA and ET-1 decreased. 6. In cells preincubated in low O2 containing medium, ET-1-stimulated [3H]-inositol phosphate accumulation was reduced by approximately 30-40%. This reduction was reversed by preincubation of the cells with Ro-318220. 7. These results suggest a role for PKC in the effects of hypoxia on PLD in pulmonary artery smooth muscle cells.