Influences of the concentration and addition time of alanine-group amino acids, i.e. alanine, leucine and valine, on serine alkaline protease (SAP) synthesis were investigated by Bacillus licheniformis (DSM 1969) carrying pHV1431::subC in a defined medium to identify the amino acids creating intracellular reaction-rate limitation in SAP production. While the precursors of alanine-group amino acids, pyruvate and alanine, did not affect SAP production considerably within the range 0-15 mM, the addition of leucine decreased both SAP production and cell formation, because of the inhibition of valine synthesis. Although valine inhibits reactions starting with pyruvate towards 2-oxo-isovalerate, due to conversion of valine into 2-oxoisovalerate and from 2-oxo-isovalerate to leucine, valine did not inhibit leucine synthesis. Val (7.5 mM) supply at t = 0 h increased SAP activity to an activity of 1070 units - cm-' which was 1.3-fold higher than that of the reference production medium. The highest cell growth yield on substrate (Y-X/S) was obtained as 0.24 g . g(-1) with the supply of alanine; and the highest product formation yield on substrate was obtained as 0.134 units . g(-1) with the supply of valine. By using the results obtained, strategies for increasing SAP production and complex medium design were also discussed.