Abstract Lipase-catalyzed synthesis of estolides starting from different saturated (C16 16OH, C18 12OH) and unsaturated (C18:1 9 cis 12-OH) hydroxy-fatty acids was investigated. For this reason, the catalytic efficiency of several native and immobilized lipases in different organic reaction media at temperatures up to 75 °C was studied. The formation of mono- and di-lactone as well as estolide’s chain elongation depends on the type and source of lipase. The lipase from Pseudomonas stutzeri immobilized by cross-linking as cross-linked enzymes aggregates (CLEAs) was the best biocatalyst in terms of chain elongation. Estolides with polymerization degree up to 10 were obtained at substrate conversions higher than 80 %.