Preparation of a single and pure phospholipid via transphosphatidylation has been a much sought after endeavor in the pharmaceutical and nonmedical industries. For this reason, phosphatidylglycerol, a lung surfactant, was produced from phosphatidylcholine with defined fatty acids, ie, dipalmitoyl phosphatidylcholine. Substrate type and concentration, enzyme source, and reaction temperature were investigated. Phospholipase D from two sources, ie, savoy cabbage, was purified in the authors' laboratory and a commercially available Streptomyces species was used for this study. The substrates used were glycerol, a polyhydric alcohol, and solketal, a monohydric form of glycerol. The progress of the reaction was monitored using thin layer chromatography, and synthesis with solketal, an unusual form of glycerol, was confirmed by liquid chromatography mass spectrometry. Surface response methodology used on four combinations of enzyme and substrate at various temperatures (30 °C-60 °C) and concentration (0.25-1 mM) revealed that yield and selectivity was temperature-driven and predictable. To validate further the thermodynamic attributes, a modified version of the Eyring equation was derived from selectivity and the Arhenius equation. These equations provide some useful insights into the difference in activation of enthalpy change(ΔΔH ++ ) and difference in activation of entropy change(ΔΔS ++ ). Plots of ln[PG]/[PA] versus 1/T gave good linear fits for these four combinations. In addition, a new thermodynamic parameter known as T PG = PA has emerged as a theoretical temperature for equivalent transferase and hydrolase activity.