Lipase from Penicillium roquefortii immobilized on porous polypropylene particles was used for enzymatic preparation of 1,2-diglycerides by alcoholysis in organic media. A screening of commercially available lipases showed that lipases fi om Penicillium species produced high amounts of 1,2-diglycerides from triglycerides. Reaction parameters such as solvent, alcohol, water activity, and fatty acid chain length were investigated. The positional selectivity of the lipase as well as the selectivity for type of glyceride specie were studied using pure isomers of the the partial glycerides. The enzyme showed high selectivity for triglycerides and 1-monoglycerides and very low activity towards diglycerides. The lipase had a clear preference for the 1- and 3-positions. The highest lipase activity was observed at low water activity, but the yield increased with increasing water activity. Above all, the regio-isomeric purity of the diglycerides increased with increasing water activity. The yield of dilaurin was 75% and furthermore, 95% of the total dilaurin was 1,2-dilaurin. Alcohol concentration and chain length of the alcohol had insignificant effect on yield and enzyme activity, but product stability increased when alcohol was present in the reaction medium. The best solvents were ethers; higher enzyme activities were obtained in aliphatic hydrocarbons but yields and regio-isomeric purities were low presumably due to acyl migration.