Enzymatic glycerolysis of soybean oil was studied. Of the nine lipases that were tested in the initial screening, Pseudomonas sp. resulted in the highest yield of monoglycerides. Lipase from Pseudomonas sp. was further studied for the influence of temperature, thermal stability, enzyme/oil ratio, and glycerol/oil ratio. A full factorial optimization approach was performed. The following conditions were tested over the specified ranges: temperature (30–70°C), thermal stability (30–70°C), enzyme/oil ratio (0.05–0.2 g enzyme/10 g oil), glycerol/oil ratio (1:1–3:1 glycerol/oil molar ratio) and 1 h reaction time. The stability of the enzyme at the reaction temperature was also incorporated as a separate variable. At temperatures above 40°C enzyme denaturation offset the higher activity. The optimal conditions were selected to be the basis for a continuous process: 40°C, a glycerol/oil molar ratio of 2:1, and an enzyme/oil ratio of 0.1 g enzyme/10 g oil. A definition for glycerolysis activity was adopted. The glycerolysis activity (1 GU) was defined as the amount of enzyme necessary to consume 1 µmol of substrate (glycerol and oil) per minute. This research is intended to explore the reaction parameters that are important in a continuous enzymatic glycerolysis process.