To examine the substrate dependency of the type 1 (PP1) and type 2A (PP2A) ser/thr protein phosphatase, the dephosphorylation of three substrates by purified preparations of PP1 and PP2A were compared. Phosphorylase kinase was used to phosphorylate glycogen phosphorylase, and protein kinase C (PKC) and cAMP- dependent protein kinase (PKA) were used to phosphorylate protamine sulfate and myelin basic protein (MBP). ³²P released from these substrates was used as a measure of phosphatase activity. Both PP1 and PP2A were active against substrates phosphorylated by phosphorylase kinase or PKC, but enzyme activity varied with substrate. In contrast, neither PP1 nor PP2A was active against substrates phosphorylated by PKA. The inhibition of PP1 activity by inhibitor-2 and inhibition of PP2A activity by okadaic acid were both substrate dependent.