Using intact pinealocytes in suspended cell culture it has been determined that acetyl CoA hydrolase activity can be rapidly increased by treatment with cystamine. Similar results are seen with diacetylcystamine, but not with GSSG, penicillamine disulfide, nor with oxidized DTT. The activation of acetyl CoA hydrolase by cystamine is reversible: after cystamine treatment is terminated, enzyme activity decreases slowly in cell culture. It is also possible to reverse the activation by treating homogenates of cystamine-treated cells with dithiothreitol. These observations are consistent with previous findings indicating that pineal acetyl CoA hydrolase activity can be regulated via protein thiol: disulfide exchange. The observations presented in this report also indicate that conditions within the cell allow this type of reaction to take place, and raise the possibility that disulfide exchange mechanisms may be physiologically involved in the intracellular regulation of the activity of this and perhaps other enzymes.