Efficiency of in-situ alkaline transesterification of dry biomass from a crop of Spirulina sp., using ultrasound-assisted and/or the addition of different co-solvents to the reaction medium was studied. The starting biomass had a total lipid content of 7.0% (Folch method), comprised mainly by the following fatty acids: 16: 0 (44.2%), 18: 3 (n-6) (23.3%), and 18: 2 (n-6) (11.1%). When the transesterification reaction was carried out without ultrasound, it only extracted 12.8% of lipids (0.9% of the biomass) after 2 h of reaction, achieving only a 63.6% ester content. When the process was carried out under the same conditions except for the ultrasound-assisted (80 W), the weight yield remained mostly unchanged, but the ester content increased to 97.6%. Additionally, when chloroform was added to methanol as a co-solvent in a 2: 1 ratio it was possible to extract 43% of lipids. However, the ester content was reduced to 69.7%. Moreover, with maximum ultrasound power (180 W) a high ester content (96.9%) and a relatively high extraction yield (26%) were obtained, even without the co-solvent. These results show that the ultrasound significant increased the yield of the in-situ alkaline transesterification of Spirulina sp. biomass, promoting a higher percent recovery of lipids as well as ester content from the starting material. (C) 2017 Elsevier Ltd. All rights reserved.