Diacylglycerol (DAG) and ceramide are important second messengers affecting cell growth, differentiation, and apoptosis. Balb/c-3T3 fibroblast cells expressing dopamine-D2S (short) receptors (Balb-D2S cells) provide a model of G-protein-mediated cell growth and transformation. In Balb-D2S cells, apomorphine (EC₅₀=10 nM) stimulated DAG and ceramide formation by 5.6- and 4.3-fold, respectively, maximal at 1 h and persisting over 6 h. These actions were blocked by pretreatment with pertussis toxin (PTX), implicating Gi/Go proteins. To address which G-proteins are involved, Balb-D2S clones expressing individual PTX-insensitive Gi proteins were treated with PTX and tested for apomorphine-induced responses. Neither PTX-insensitive Gi2 nor Gi3 rescued D2S-induced DAG or ceramide formation. Both D2S-induced DAG and ceramide signals required G subunits and were blocked by inhibitors of phospholipase C (U73,122 and partially by D609). The similar G-protein specificity of D2S-induced calcium mobilization, DAG and ceramide formation indicates a G-dependent phospholipase C-mediated pathway to increase DAG and ceramide formation. Both D2 agonists and ceramide specifically induced mitogen-activated protein kinase (ERK1/2), suggesting that ceramide mediates a novel pathway of D2-induced ERK1/2 activation leading to cell growth.