We have used a combination of highly specific protein phosphatase Inhibitors and purified mammalian protein phosphatases to show that at least two separate Ser/Thr protein phosphatase activities are required for pre-mRNA splicing, but not for spllceosome assembly. Okadaic acid, tautomycin, and mlcrocystin-LR, which are potent and specific inhibitors of PP1 and PP2A, two of the four major types of Ser/Thr-specific phosphatase catalytic subunits, block both catalytic steps of the pre-mRNA splicing mechanism in HeLa nuclear extracts. Inhibition of PP2A inhibits the second step of splicing predominantly while Inhibition of both PP1 and PP2A blocks both steps, indicating a differential contribution of PP1 and PP2A activities to the two separate catalytic steps of splicing. Splicing activity is restored to toxin-inhibited extracts by the addition of highly purified mammalian PP1 or PP2A. Protein phosphatase activity was not required for efficient assembly of splicing complexes containing each of the U1, U2, U4/U6 and U5 snRNPs. The data indicate that reversible protein phosphorylation may play an Important role in regulating the pre-mRNA splicing mechanism.