The phospholipid polar headgroup composition of membranes of C6 cells was modified in vivo by growth for 24h in media supplemented with the polar headgroup precursors, N, N'-dimethylethanolamine, N-monomethylethanolamine or ethanolamine. These modifications were achieved without alteration of the cholesterol and phospholipid content of the membranes, and without changes in the fatty acyl or protein composition of these membranes. No changes were found in the physical properties of membranes isolated from these cells . Enriching the cell membranes with PDME, PNME or PE elevated the basal intracellular cAMP content, but decreased the degree of stimulation of intracellular cAMP content in response to β-adrenergic stimulation. This reduction of the β-adrenergic response appeared to be due to enhanced phosphodiesterase activity. β-adrenergic stimulation did not affect bulk physical properties of the membrane. Exposure to isoproterenol increased the amount of [3H] methyl label recovered from TLC plates but the different distribution of label indicated that such analysis is insufficient to demonstrate specific phospholipid methylation. This system is a useful means of studying the relationship between phospholipid composition, phospholipid metabolism and the β-adrenergic response in the C6 cell line.