The conversion of 1-acyl-sn-glycero-3-phosphorylinositol-3H into phosphatidylinositol-3H was studied using rat liver microsomal and homogenate preparations. The nature of the molecular species of phosphatidyl inositol so formed in the absence of added acyl moieties was determined after fractionating the radioactive product by means of argentation thin layer chromatography. In other experiments, the possible specificity of the microsomal acyl-CoA:1-acyl-sn-glycero-3-phosphorylinositol acyltransferase towards different acyl-CoA derivatives was investigated. Maximum conversion of 1-acyl GPI to the diacyl analogue was dependent on the addition of adenosine triphosphate and CoA when exogensou acyl groups were omitted from the incubation medium. Under these latter conditions, the tetraenoic species comprised 56-74% of the total molecular species of newly-formed phosphatidylinositol. The microsomal acyl-CoA:1-acyl-sn-glycero-3-phosphorylinositol acyltransferase showed a marked preference for arachidonoyl-CoA. The present results suggest that the enrichment of rat liver phosphatidyl inositol in arachidonic acid may arise when 1-acyl-sn-glycero-3-phosphorylinositol is acylated to form phosphatidylinositol.