A new procedure has been developed for the large scale preparation of recombinant 2-deoxyribose-5-phosphate aldolase (DERA, EC 4.1.2.4) from E. coli strain DH5 alpha (ATCC 86963). The enzyme was purified to homogeneity with an overall yield of 83% and in sufficient quantity to grow crystals suitable for X-ray diffraction studies. Using the sequential two- or three-substrate aldol reaction, DERA was applied to the synthesis of a variety of sugar analogs including deoxyriboses, 2-deoxyfucose analogs, dideoxyhexoses, trideoxyhexoses, deoxythiosugars, and C-13-substituted 2-deoxyribose-5-phosphate.