Pseudomonas sp. F12 isolated from soil could transform dl-2-amino-Delta(2)-thiazoline-4-carboxylic acid (DL-ATC) to l-cysteine. It could grow in minimal medium containing DL-ATC as the sole carbon and nitrogen source, and the apparent activity of l-cysteine synthesis (CS) achieved 122 U/mL in a 5-L bioreactor. Pseudomonas sp. F12 could utilize glucose as carbon source and ammonia as nitrogen source for growth, but no CS activity was formed. To reduce the cost of DL-ATC, the cultivation process was divided into a growth stage on glucose and ammonia and a production stage induced by DL-ATC. The excessive glucose led to the production of byproduct(s) which seriously inhibited cell growth and CS production. Ammonium was accumulated when DL-ATC was consumed, and ammonium did not inhibit CS activity formation until 60 mM. Based on the above features, fed-batch cultivation of the growth stage was developed by supplying glucose restrictively. The volumetric CS activity was enhanced more than two times that obtained under the initial conditions.