Microbial lipases are of particular interest for biotechnological applications because of their high specificity and their ability to perform reactions at room temperature. Here are characterized extracellular and cell-bound crude lipase extracts from the yeasts Pseudozyma tsukubaensis CBS 422.96, Candida rugosa CBS 5213, and Geotrichum candidum NRRL Y-552. Enzyme preparations were optimally active at neutral pH and ambient temperature, and displayed different substrate specificities. Unlike P. tsukubaensis and C. rugosa extracts, crude lipases from G. candidum were unable to hydrolyze ethyl esters of saturated fatty acids (C8:0–C18:0). The activity of extracts from P. tsukubaensis was maximal for the octyl chain and then decreases as the acyl chain was lengthened, while cell-bound lipases from C. rugosa hydrolyzed any saturated fatty acid ethyl esters from C8:0 to C18:0. Preparations from C. rugosa and P. tsukubaensis displayed a sn-1,3-regioselectivity on triolein, while cell-bound lipases from G. candidum were non-regioselective. The extracellular crude lipases from G. candidum exhibited a small preference for the internal position both on triolein and on six edible oils, releasing a high proportion of DAG-1,3 (~50%) compared to DAG-1,2(2,3). To the contrary, extracts from P. tsukubaensis and C. rugosa catalyzed the formation of almost 100% DAG-1,2(2,3) from oils.