Thiols are important molecules to control apoptosis. This study examined the effect of N-acetyl-L-cysteine (NAC) on in vitro spontaneous apoptosis of human tonsillar B lymphocytes (TBL). Results show that NAC inhibits TBL apoptosis and maintains their survival in vitro. The antiapoptotic action of NAC is progressively reduced when its addition to culture is delayed, is reversible, and is not blocked by cycloheximide. The antiapoptotic activity of NAC is associated with its ability to inhibit caspase-3 and -7 proteolytic processing, DNA-fragmentation factor 45 cleavage, and DNA fragmentation. Furthermore, NAC inhibits BID cleavage and cytochrome c release from mitochondria and increases the expression of Bcl-2 and Bcl(XL) survival proteins. However, it has no effect on caspase-9 cleavage and increases that of caspase-8 and poly(adenosine 5'-diphosphate-ribose)polymerase. We conclude that NAC-induced inhibition of TBL apoptosis is associated with inhibition of caspase-3 and -7 processing and is accompanied by changes in several regulatory components of the apoptotic process. These results pose the question of whether microenvironment thiols may in part contribute to in vivo B cell survival.