Many protein kinases are able to catalyze the thiophosphorylation of protein substrates via a phospho-transfer reaction using adenosine-5'-o(3-thiotriphosphate) (ATPgammaS) (1), but, in general, thiophosphorylated proteins are very poor substrates for protein phosphatases (2,3). As a result, the protein substrate is essentially trapped in the phosphorylated state. This thiophosphorylation can be exploited in order to generate a strategy for the selective purification of protein phosphatases. Indeed, a number of thiophosphorylated protein substrates have been successfully used for the affinity purification of protein phosphatases (4-7). Here we describe the use of thiophosphorylated smooth muscle myosin regulatory light chains for the selective purification of the smooth muscle myosin phosphatase holoenzyme.