The study of triacylglycerol synthesis using 1,3-regiospecific-immobilized Rhizomucor miehei lipase as biocatalyst was performed under pure substrate conditions. Tritetradecanoylglycerol, trihexadecanoylglycerol, and trioctadecanoylglycerol were synthesized from glycerol and free fatty acids at a higher rate than medium-chain triacylglycerols such as tridecanoylglycerol and tridodecanoylglycerol. Furthermore, it is clearly shown that linolenic acid is esterified more slowly than oleic acid which is itself esterified moo-e slowly than octadecanoic acid. The higher the number of unsaturation, the lower the rate of synthesis and the final yield Under the tested conditions, trilinolenin synthesis cannot be achieved because secondary reactions such as polymerization took place.