The influence of HDL3 on phospholipid breakdown was examined in human skin fibroblasts. HDL3 elicited phosphatidylcholine (PC) and phosphatidylinositol (PI) turnover and activated multiple phospholipases. In [14C]lyso-PC-labeled or [14C]choline (Cho)-labeled cells, a biphasic activation of PC-specific phospholipase D (PLD) with peak maxima 30 to 60 seconds and 5 to 7 minutes after stimulation with 20 micrograms/mL HDL3 was shown by (1) a 1.5- to 3-fold increase in Cho release, and (3) transphosphatidylation of PC to phosphatidylbutanol in the presence of 0.3% butanol. Activation of PC-specific PLD was paralleled by an activation of PC-specific phospholipase C (PLC). A significant increase in [14C]diacylglycerol (DG) was seen from 2 minutes after stimulation onward and remained for at least 2 hours. By means of butanol, the PA-phosphohydrolase (PPH) inhibitor propranolol, and the PC-PLC inhibitor D609, we demonstrated that the initial PC-derived DG formation occurred primarily by a coupled PLD/PPH pathway and that a major part of the sustained DG formation was derived directly from PC by PC-PLC. By down-regulating protein kinase C (PKC) we demonstrated that PKC activates PC-PLC and desensitizes PC-PLD at no longer incubation times. The sustained PC hydrolysis as well as HDL3-mediated PI turnover and PC resynthesis was observed on stimulation with 5 to 75 micrograms/mL HDL3, whereas the rapid activation of PC-PLD/PPH was detected only on stimulation with HDL3 at concentrations of between 10 and 75 micrograms/mL. Only the latter response could be mimicked by apolipoprotein A-I and apolipoprotein A-II proteoliposomes, and only this response was inducible by cholesterol loading. The HDL3-mediated second-messenger responses were inhibited by modification of HDL3 by tetranitromethane and could not be mimicked by protein-free liposomes. These data suggest that HDL3-induced cell signaling in human skin fibroblasts is mediated by specific protein-receptor interaction and that more than one agonist activity may be involved.