A novel isomer of phosphatidylinositol that differs in the structure of the head group was detected in barley (Hordeurn vulgate cv Himalaya) seeds. In this paper we describe our efforts to elucidate the structure of the novel isomer. Evidence from a variety of techniques, including chemical modification of in vivo 32Pi- and myo-[3H]inositol-labeled compounds, gas chromatography-mass spectrometry analysis, in vivo incorporation of scyllo-[3H]inositol, and enzymatic studies that suggest that the structure is phosphatidylscyllo-inositol (scyllo-PI), is presented. The use of microwave energy to significantly enhance the slow rate of hydrolysis of phosphoinositides is described. The presence of scyllo-PI can be easily overlooked by the methods commonly employed; therefore, experimental considerations important for the detection of scyllo-PI are discussed.