In this work, density functional calculations were employed to revisit the formation mechanism of enamine, the intermediate to mediate the proline-catalyzed direct aldol reaction. Different from the work of Boyd et al. [24], the plausible reaction routes were calculated on basis of the complete conformational studies of proline and acetone interacted complexes. Six energy minima were determined and their relative stabilities increase in the order PAf < PAe < PAb < PAc < PAa < Pad. These structures are mainly stabilized through the hydrogen bonds of the proline-H atoms and the acetone-O atoms. Routes 2 and 4 are the most probable to take place. The rate-determining step of Route 2 is the formation of C3-N bond with the energy barrier equal to 86 kJ mol(-1). This step is accelerated by the following barrierless process. As the theoretical results indicated, Route 4 needs to start with at least 2 mole equiv. acetone, consistent with the high acetone concentrations used in experiments. The rate-determining step of Route 4 is the intra-carboxyl proton transfer. which was revealed to be greatly assisted by solvents. (C) 2009 Elsevier B.V. All rights reserved.