The neural substrates of learning and memory most likely involve activity-dependent long-term changes in synaptic strength, including long-term potentiation and long-term depression. A critical element in the cascade of events hypothesized to underlie such changes in synaptic function is modification of protein phosphorylation. Long-term depression is thought to involve decreases in protein phosphorylation, which could result from reduction in protein kinase activity and/or enhancement in protein phosphatase activity. We present here direct evidence that long-term depression in the hippocampus in vivo is associated with an increase in the activity of the serine/threonine phosphatases 1 and 2A. The increase in activity of phosphatase 1 was transient, whereas that of phosphatase 2A lasted > 65 min after the induction of long-term depression. Blockade of long-term depression prevented the observed increases in phosphatase activity, as did selective inhibition of phosphatase 1 and 2A. Induction of long-term depression had no effect on the level of either phosphatase, which suggests that our results reflect increases in the intrinsic activity of these two enzymes. Our findings are consistent with a model of synaptic plasticity that implicates protein dephosphorylation by serine/threonine phosphatases in the early maintenance and/or expression of long-term depression of synaptic strength.