Systematic screening of secretion proteins using an approach based on the completely sequenced genome of Corynebacterium glutamicum R revealed 405 candidate signal peptides, 108 of which were able to heterologously secrete an active-form alpha-amylase derived from Geobacillus stearothermophilus. These comprised 90 general secretory (Sec)-type, 10 twin-arginine translocator (Tat)-type and eight Sec-type with presumptive lipobox peptides. Only Sec- and Tat-type signals directed high-efficiency secretion. In two assays, 11 of these signals resulted in 50- to 150-fold increased amounts of secreted alpha-amylase compared with the well-known corynebacterial secretory protein PS2. While the presence of an AXA motif at the cleavage sites was readily apparent, it was the presence of a glutamine residue adjacent to the cleavage site that may affect secretion efficiency.