Mechanical forces applied to cultured bone cells induce the production of cAMP via stimulation of the formation of prostaglandin E₂ (PGE₂) and its release into the medium, resulting in stimulation of adenylate cyclase. In this paper we show that either the antibiotic gentamycin (100 μg/ml) or antiphospholipid antibodies (0.1%) which bind to membrane phospholipids abolish cAMP formation induced by mechanical forces; exogenously added arachidonic acid or PGE₂ stimulates cAMP formation, even in the presence of these agents. Addition of exogenous phospholipase A₂ (but not phospholipase C) causes an increase in the formation of cAMP in bone cells, a response that is also inhibited by gentamycin or antiphospholipase antibodies. These observations suggest that mechanical forces exert their effect on bone cells via the following chain of events: (1) activation of phospholipase A₂, (2) release of arachidonic acid, (3) increased PGE synthesis, (4) augmented cAMP production.